141 Chapter 3

[1 2-a] pyridin-3-yl] methanol

Imp-5

Imp-7

32 Literature Search

A thorough literature search revealed that only a few HPLC methods had

been discussed for the determination of ZT in pharmaceutical preparations [7ndash

10] Quantifications of ZT by liquid chromatographic methods have been

officially published in USP and EP [11-12] A potentiometric method was

reported for the estimation of ZT in human serum and tablet formulation [13]

determination of ZT tablets by means of spectrophotometric methods had been

published [14-15] Two methods describing the testing of zolpidem in oral fluid

using Liquid chromatographyndashtandem mass spectrometric methods have been

reported [16-17] HPLC method pertaining to chromatographic behavior of ZT

and its degradation products has been published [18]

Only a few stability-indicating HPLC methods are reported in the literature

The official monograph methods are not capable of separating the potential

impurities and degradation products The existing isocratic HPLC methods

cannot give adequate resolution between Imp-5 amp Imp-6 Furthermore the

approximate run time is 35 min According to our extensive survey there is no

single analytical method reported in the literature that is capable of separating

all the potential impurities and degradation products of ZT in drug substance

and drug product at shorter run times

142 Chapter 3

33 Ultra Performance Liquid Chromatography (UPLC)

Ultra-performance liquid chromatography (UPLC) is a new class of

separation technique based upon deep-rooted principles of liquid

chromatography which utilizes sub-2microm particles for column stationary phase

(CSP) These particles function at elevated mobile phase linear velocities

resulting in an amazing increase in resolution sensitivity and speed of

analysis

The increase in the number of impurities in the drug poses challenges in

regular chromatographic method development while achieving adequate

resolutions at shorter run times However recent advances in the use of LC

technology have significantly facilitated progress to prevail over these

challenges UPLC is such an advancement that accentuates chromatographic

high throughput in terms of speed sensitivity and resolution Because of these

attributes UPLC has gained immense importance in pharmaceutical analysis

Hence the present work is aimed at developing a selective fast cost-effective

and stability indicating method using the contemporary UPLC technique and

subsequently the method is validated as per ICH guidelines [19]

34 pH Gradient

A theory of pH gradient has been applied in the current work A

programmed increase of eluent strength during chromatographic procedure by

altering mobile phase pH is known in the prior art [20-21] A narrow range

143 Chapter 3

pH gradient together with organic modifier has been used in the present UPLC

method to achieve separation between the critical pairs

35 Experimental

351 Chemicals and Reagents

Zolpidem tartrate and its related compounds were obtained from Dr

Reddyrsquos Laboratories Limited IPDO Hyderabad India Commercially available

brand Ambienreg tablets were used for dosage form analysis Analytical grade

sodium dihydrogen ortho phosphate monohydrate (NaH2PO4H2O) ortho-

phosphoric acid (H3PO4) HPLC-grade solvents ie methanol (MeOH) and

acetonitrile (ACN) were procured from Merck Darmstadt Germany

Triethylamine (TEA) was bought from JT Baker Mallinckrodt Inc Phillipsburg

NJ USA High purity water has been prepared by using Millipore Milli Q plus

water purification equipment (Millipore Corporate Headquarters Billerica MA)

352 Chromatographic Conditions and Equipment

Liquid chromatographic analysis was performed on Waters Acquity UPLC

equipped with photodiode array detector (PDA) at a wavelength of 254 nm using

Empower-2 software Separation was achieved on C18 column ie Acquity

UPLC HSS T3-C18 of dimensions 100 x 21mm 18microm at a flow rate of 03

mLmin using a gradient program (TB) set as 0015 255 3560 5060

8270 855 and 1005 Buffer solution was prepared by adding 1mL of

Triethyl amine (TEA) to 1000mL of 10 mM NaH2PO4 H2O Mobile phase A was a

mixture of buffer (pH adjusted to 55 with H3PO4) ACN and MeOH in the ratio

of 602020 (vvv) Mobile phase B was a mixture of buffer (pH adjusted to 73

144 Chapter 3

with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature

was maintained at 25ordmC The injection volume was 10 microL

353 LC-MSMS conditions

An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled

with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with

Analyst 14 software MDS SCIEX USA) was utilized for mass identification of

degradation products A Novapak C18 column with dimensions 150 x 39 mm

4μm was employed as a stationary phase An ammonium acetate (Merck

Darmstadt Germany) solution of concentration 002 mM was used as buffer A

mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was

used as mobile phase The column oven temperature was maintained at 25degC

ACN was used as diluent The flow rate was set as 15 mLmin The analysis

was carried out in positive electro-spraypositive ionization mode the ion

source voltage was 5000 V and the source temperature was 450degC GS1 and

GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20

psi

36 Preparation of solutions

361 Preparation of Stock solution

A stock solution of ZT of concentration 500 microgmL was prepared by

dissolving an appropriate quantity of the drug in a diluent containing a mixture

of acetonitrile and water in the ratio of 8020 (vv) Working solutions of

concentrations 50microgmL and 5microgmL were prepared from this stock solution

for the determination of related compounds and assay respectively Composite

145 Chapter 3

and individual stock solutions of impurities having concentration 05microgmL

each were prepared in the diluent

362 Preparation of sample solution

Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average

tablet weight was determined These tablets were transferred into a clean dry

mortar and grounded into fine powder The fine powdered sample equivalent to

50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH

Buffer 7030 vv) to make a solution of concentration 500microgmL The solution

was subjected to sonication for 30min and mechanical shaking for each 30

minutes The solution was filtered from which a 10mL of filtered solution was

diluted to 10 mL with the aid of mobile phase A The resulting solution of

concentration 50microgmL was filtered through 022μm nylon membrane filter

The solution was then analyzed in UPLC for related substances analysis The

solution was further diluted to obtain the concentration of 5microgmL for assay

determination Similar concentration solutions were prepared for API analysis

363 Generation of Stress sample solutions

A qualified sample of ZT has been chosen to conduct the stress study

According to ICH stability guidelines (Q1AR2) stress studies are likely to be

performed to study the intrinsic stability of the molecule ZT has been exposed

to various stress conditions such as heat light acid (HCl) base (NaOH)

oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated

below

Table 31 Stress study conditions for ZT

146 Chapter 3

37 Method development

371 Objectives of method development

1 Rapid separation of ZT and its eight potential impurities

2 Identification of possible degradation products by means of LCMS analysis

and evaluation of peak purity

SNo Stressed Agent Stressing Condition

1 Heat

API and tablets were exposed to dry heat

at 105degC for about 7 days

2 Light API and tablets were exposed to UV light

at 254nm for 7 days

3 Acid (5N HCl) API and tablets extracted solutions were

treated with 5N HCl 60degC for 24 hrs

4 Base (5N NaOH)

API and tablets solutions were treated

with 5N NaOH at 60degC for 90mins

5 Oxidation (50 H2O2) API and tablets solutions were treated

with 5 H2O2 at 60degC for 2 hrs

6 Water hydrolysis API and tablets solutions were treated

with water at 60degC for 24h

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

143 Chapter 3

pH gradient together with organic modifier has been used in the present UPLC

method to achieve separation between the critical pairs

35 Experimental

351 Chemicals and Reagents

Zolpidem tartrate and its related compounds were obtained from Dr

Reddyrsquos Laboratories Limited IPDO Hyderabad India Commercially available

brand Ambienreg tablets were used for dosage form analysis Analytical grade

sodium dihydrogen ortho phosphate monohydrate (NaH2PO4H2O) ortho-

phosphoric acid (H3PO4) HPLC-grade solvents ie methanol (MeOH) and

acetonitrile (ACN) were procured from Merck Darmstadt Germany

Triethylamine (TEA) was bought from JT Baker Mallinckrodt Inc Phillipsburg

NJ USA High purity water has been prepared by using Millipore Milli Q plus

water purification equipment (Millipore Corporate Headquarters Billerica MA)

352 Chromatographic Conditions and Equipment

Liquid chromatographic analysis was performed on Waters Acquity UPLC

equipped with photodiode array detector (PDA) at a wavelength of 254 nm using

Empower-2 software Separation was achieved on C18 column ie Acquity

UPLC HSS T3-C18 of dimensions 100 x 21mm 18microm at a flow rate of 03

mLmin using a gradient program (TB) set as 0015 255 3560 5060

8270 855 and 1005 Buffer solution was prepared by adding 1mL of

Triethyl amine (TEA) to 1000mL of 10 mM NaH2PO4 H2O Mobile phase A was a

mixture of buffer (pH adjusted to 55 with H3PO4) ACN and MeOH in the ratio

of 602020 (vvv) Mobile phase B was a mixture of buffer (pH adjusted to 73

144 Chapter 3

with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature

was maintained at 25ordmC The injection volume was 10 microL

353 LC-MSMS conditions

An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled

with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with

Analyst 14 software MDS SCIEX USA) was utilized for mass identification of

degradation products A Novapak C18 column with dimensions 150 x 39 mm

4μm was employed as a stationary phase An ammonium acetate (Merck

Darmstadt Germany) solution of concentration 002 mM was used as buffer A

mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was

used as mobile phase The column oven temperature was maintained at 25degC

ACN was used as diluent The flow rate was set as 15 mLmin The analysis

was carried out in positive electro-spraypositive ionization mode the ion

source voltage was 5000 V and the source temperature was 450degC GS1 and

GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20

psi

36 Preparation of solutions

361 Preparation of Stock solution

A stock solution of ZT of concentration 500 microgmL was prepared by

dissolving an appropriate quantity of the drug in a diluent containing a mixture

of acetonitrile and water in the ratio of 8020 (vv) Working solutions of

concentrations 50microgmL and 5microgmL were prepared from this stock solution

for the determination of related compounds and assay respectively Composite

145 Chapter 3

and individual stock solutions of impurities having concentration 05microgmL

each were prepared in the diluent

362 Preparation of sample solution

Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average

tablet weight was determined These tablets were transferred into a clean dry

mortar and grounded into fine powder The fine powdered sample equivalent to

50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH

Buffer 7030 vv) to make a solution of concentration 500microgmL The solution

was subjected to sonication for 30min and mechanical shaking for each 30

minutes The solution was filtered from which a 10mL of filtered solution was

diluted to 10 mL with the aid of mobile phase A The resulting solution of

concentration 50microgmL was filtered through 022μm nylon membrane filter

The solution was then analyzed in UPLC for related substances analysis The

solution was further diluted to obtain the concentration of 5microgmL for assay

determination Similar concentration solutions were prepared for API analysis

363 Generation of Stress sample solutions

A qualified sample of ZT has been chosen to conduct the stress study

According to ICH stability guidelines (Q1AR2) stress studies are likely to be

performed to study the intrinsic stability of the molecule ZT has been exposed

to various stress conditions such as heat light acid (HCl) base (NaOH)

oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated

below

Table 31 Stress study conditions for ZT

146 Chapter 3

37 Method development

371 Objectives of method development

1 Rapid separation of ZT and its eight potential impurities

2 Identification of possible degradation products by means of LCMS analysis

and evaluation of peak purity

SNo Stressed Agent Stressing Condition

1 Heat

API and tablets were exposed to dry heat

at 105degC for about 7 days

2 Light API and tablets were exposed to UV light

at 254nm for 7 days

3 Acid (5N HCl) API and tablets extracted solutions were

treated with 5N HCl 60degC for 24 hrs

4 Base (5N NaOH)

API and tablets solutions were treated

with 5N NaOH at 60degC for 90mins

5 Oxidation (50 H2O2) API and tablets solutions were treated

with 5 H2O2 at 60degC for 2 hrs

6 Water hydrolysis API and tablets solutions were treated

with water at 60degC for 24h

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

144 Chapter 3

with H3PO4) and ACN in the ratio of 4555 (vv) The column oven temperature

was maintained at 25ordmC The injection volume was 10 microL

353 LC-MSMS conditions

An LCndashMSMS system (Agilent 1100 series liquid chromatograph coupled

with Applied Biosystems 4000 Q Trap triple quadrupole mass spectrometer with

Analyst 14 software MDS SCIEX USA) was utilized for mass identification of

degradation products A Novapak C18 column with dimensions 150 x 39 mm

4μm was employed as a stationary phase An ammonium acetate (Merck

Darmstadt Germany) solution of concentration 002 mM was used as buffer A

mixture of Ammonium acetate buffer MeOH and ACN (590230180 vvv) was

used as mobile phase The column oven temperature was maintained at 25degC

ACN was used as diluent The flow rate was set as 15 mLmin The analysis

was carried out in positive electro-spraypositive ionization mode the ion

source voltage was 5000 V and the source temperature was 450degC GS1 and

GS2 were set to 30 and 35 psi respectively The curtain gas flow was set at 20

psi

36 Preparation of solutions

361 Preparation of Stock solution

A stock solution of ZT of concentration 500 microgmL was prepared by

dissolving an appropriate quantity of the drug in a diluent containing a mixture

of acetonitrile and water in the ratio of 8020 (vv) Working solutions of

concentrations 50microgmL and 5microgmL were prepared from this stock solution

for the determination of related compounds and assay respectively Composite

145 Chapter 3

and individual stock solutions of impurities having concentration 05microgmL

each were prepared in the diluent

362 Preparation of sample solution

Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average

tablet weight was determined These tablets were transferred into a clean dry

mortar and grounded into fine powder The fine powdered sample equivalent to

50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH

Buffer 7030 vv) to make a solution of concentration 500microgmL The solution

was subjected to sonication for 30min and mechanical shaking for each 30

minutes The solution was filtered from which a 10mL of filtered solution was

diluted to 10 mL with the aid of mobile phase A The resulting solution of

concentration 50microgmL was filtered through 022μm nylon membrane filter

The solution was then analyzed in UPLC for related substances analysis The

solution was further diluted to obtain the concentration of 5microgmL for assay

determination Similar concentration solutions were prepared for API analysis

363 Generation of Stress sample solutions

A qualified sample of ZT has been chosen to conduct the stress study

According to ICH stability guidelines (Q1AR2) stress studies are likely to be

performed to study the intrinsic stability of the molecule ZT has been exposed

to various stress conditions such as heat light acid (HCl) base (NaOH)

oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated

below

Table 31 Stress study conditions for ZT

146 Chapter 3

37 Method development

371 Objectives of method development

1 Rapid separation of ZT and its eight potential impurities

2 Identification of possible degradation products by means of LCMS analysis

and evaluation of peak purity

SNo Stressed Agent Stressing Condition

1 Heat

API and tablets were exposed to dry heat

at 105degC for about 7 days

2 Light API and tablets were exposed to UV light

at 254nm for 7 days

3 Acid (5N HCl) API and tablets extracted solutions were

treated with 5N HCl 60degC for 24 hrs

4 Base (5N NaOH)

API and tablets solutions were treated

with 5N NaOH at 60degC for 90mins

5 Oxidation (50 H2O2) API and tablets solutions were treated

with 5 H2O2 at 60degC for 2 hrs

6 Water hydrolysis API and tablets solutions were treated

with water at 60degC for 24h

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

145 Chapter 3

and individual stock solutions of impurities having concentration 05microgmL

each were prepared in the diluent

362 Preparation of sample solution

Brand Ambienreg 10 mg of quantity 20 tablets was weighed and the average

tablet weight was determined These tablets were transferred into a clean dry

mortar and grounded into fine powder The fine powdered sample equivalent to

50 mg of the drug was weighed and dissolved in 100 mL of diluent (MeOH

Buffer 7030 vv) to make a solution of concentration 500microgmL The solution

was subjected to sonication for 30min and mechanical shaking for each 30

minutes The solution was filtered from which a 10mL of filtered solution was

diluted to 10 mL with the aid of mobile phase A The resulting solution of

concentration 50microgmL was filtered through 022μm nylon membrane filter

The solution was then analyzed in UPLC for related substances analysis The

solution was further diluted to obtain the concentration of 5microgmL for assay

determination Similar concentration solutions were prepared for API analysis

363 Generation of Stress sample solutions

A qualified sample of ZT has been chosen to conduct the stress study

According to ICH stability guidelines (Q1AR2) stress studies are likely to be

performed to study the intrinsic stability of the molecule ZT has been exposed

to various stress conditions such as heat light acid (HCl) base (NaOH)

oxidative (H2O2) and water hydrolysis The final stress conditions were tabulated

below

Table 31 Stress study conditions for ZT

146 Chapter 3

37 Method development

371 Objectives of method development

1 Rapid separation of ZT and its eight potential impurities

2 Identification of possible degradation products by means of LCMS analysis

and evaluation of peak purity

SNo Stressed Agent Stressing Condition

1 Heat

API and tablets were exposed to dry heat

at 105degC for about 7 days

2 Light API and tablets were exposed to UV light

at 254nm for 7 days

3 Acid (5N HCl) API and tablets extracted solutions were

treated with 5N HCl 60degC for 24 hrs

4 Base (5N NaOH)

API and tablets solutions were treated

with 5N NaOH at 60degC for 90mins

5 Oxidation (50 H2O2) API and tablets solutions were treated

with 5 H2O2 at 60degC for 2 hrs

6 Water hydrolysis API and tablets solutions were treated

with water at 60degC for 24h

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

146 Chapter 3

37 Method development

371 Objectives of method development

1 Rapid separation of ZT and its eight potential impurities

2 Identification of possible degradation products by means of LCMS analysis

and evaluation of peak purity

SNo Stressed Agent Stressing Condition

1 Heat

API and tablets were exposed to dry heat

at 105degC for about 7 days

2 Light API and tablets were exposed to UV light

at 254nm for 7 days

3 Acid (5N HCl) API and tablets extracted solutions were

treated with 5N HCl 60degC for 24 hrs

4 Base (5N NaOH)

API and tablets solutions were treated

with 5N NaOH at 60degC for 90mins

5 Oxidation (50 H2O2) API and tablets solutions were treated

with 5 H2O2 at 60degC for 2 hrs

6 Water hydrolysis API and tablets solutions were treated

with water at 60degC for 24h

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

147 Chapter 3

3 Single analytical method for the determination of assay and related

substances in bulk actives and dosage forms

4 Targeted for a minimum resolution of 15 between impurities and tailing

factor lt 20 for ZT peak at assay method

5 Aimed at LOQ values ie 0375microgmL (50 of the specification level which

is equivalent to 0375microgmL) for all the impurities of ZT

372 Method development strategy

A systematic method development approach had been followed to achieve

successful separation The key steps are mentioned below

Fig 33 Flow diagram of method development strategy

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

148 Chapter 3

373 Classification of the sample

Zolpidem tartrate and its properties

1 Chemical name N N 6-trimethyl-2-p-tolylimidazo [1 2-a] pyridine-3-

acetamide L-(+)-tartrate (21)

2 Chemical structure The polar groups in the structure are highlighted in

circles

3 Molecular weight 76488 as tartrate salt

30739 as base

4 Solubility Slightly soluble in water and sparingly soluble in

alcohol

5 pKa 62

6 Log P 12

7 Nature of the

molecule

Basic in nature and ionic compound

8 UV sensitivity

UV active since ZT has phenyl ring systems with

extended conjugated double bonds in its chemical

structure

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

149 Chapter 3

374 Impurity details

Zolpidem tartrate has eight potential impurities as mentioned in the section

31 The source and classifications of the impurities are tabulated below Table

32

Table 32 Origin of impurities for ZT

SNo Name of the impurity Source of impurity

Process related Degradation related

1 Imp-1 No Yes

2 Imp-2 Yes Yes

3 Imp-3 Yes No

4 Imp-4 Yes Yes

5 Imp-5 Yes No

6 Imp-6 Yes No

7 Imp-7 Yes No

8 Imp-8 Yes No

375 Preliminary chromatographic conditions

3751 Selection of detection wavelength

A composite solution containing 50 μgmL of drug and 1 μgmL of each of

the eight impurities was prepared in the diluent All the samples were analyzed

in HPLC- PDA system and the UV spectrums for all the components were

extracted

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

150 Chapter 3

Fig 34 Individual and overlaid UV spectra of ZT and its impurities

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

151 Chapter 3

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

152 Chapter 3

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

153 Chapter 3

Selection of wavelength to monitor zolpidem tartrate and its impurities

An optimal wavelength of detection for ZT and its impurities was selected as

254 nm based on UV spectra using PDA detector for LC analysis Fig 34

3752 Selection of UPLC column

Initial experiments were carried out on different commercially available RP-

UPLC columns such as Acquity UPLC BEH C18 (17microm 21x 100 mm) BEH

shield RP 18 (17microm 21x 100 mm) and HSS T3-C18 (18microm 21x 100 mm) to

determine the selectivity and symmetry of the individual components In all the

columns cited above Imp-1 amp Imp-2 peaks were closely eluted with baseline

separation and imp-5 amp imp-6 peaks were merged The remaining peaks were

separated with resolution (RS)gt15 and the peak tailing was greater than 15 for

ZT Due to the structural similarities co-elution of above mentioned impurity

0970 Imp-1

1134 Imp-2

1978 Imp-3

2486 Imp-4

3443 Imp-5

3619 Imp-6

3897 ZT

4796 Imp-7

5671 Imp-8

2631

3438

20672114

3200

3578

3057

3710

2114

2443

3854

2443

3105

2114

2443

3057

nm

22000 24000 26000 28000 30000 32000 34000 36000 38000

mA

u

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

154 Chapter 3

pairs was observed Additionally peak tailing was also observed due to the

secondary interactions with residual silanol groups Although separation was

similar in all the three columns HSS T3 column had shown relatively good

peak symmetry and efficiency so it was thus selected for further optimization

Addition of triethylamine in the buffer improved the peak shape of ZT but the

resolution between imp-5 amp imp-6 remained critical

3753 Effect of buffer pH

The important parameters buffer pH and gradient mode of elution that

would likely to have significant effect on critical pair resolution were

investigated There was no substantial change in the resolution between Imps-1

amp 2 (Rs1) when the buffer pH increased to 55 furthermore RS1 slightly

decreased at pH 70 (Table 33) Conversely the resolutions between Imp-5 amp

6 (RS2) and Imp-6 amp ZT (RS

3) were improved when the pH altered from 60 to 70

The increase in Rs1 in acidic mobile phase and RS

2 RS3 in basic mobile phase

can be explained by the fact that acidic analytes (Imp-1amp Imp-2) in buffers of

adequately low pH will remain unionized and get increased retentions On the

other hand neutral basic compounds (Imp-5 Imp-6ampZT) at higher pH will be

more retained From table 33 acceptable resolutions were obtained at pH 70

with isocratic elution and pH 75 with gradient elution but in both the cases

the run time was longer

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

155 Chapter 3

Table 33 Effect of Buffer pH on resolution of critical pairs

Mode of

elution Buffer pH (Rs1)a (Rs2)b (Rs3)c Run time

Isocratic 40 21 lt10 17 15min

Isocratic 50 22 lt10 17 14min

Isocratic 60 21 11 18 15min

Isocratic 70 20 18 22 gt15min

Gradient 55 21 lt10 17 8min

Gradient 75 18 22 23 gt15min

Gradient A 55 B

73 25 21 22 8 min

a Rs1 Resolution between Imp-1 and Imp-2

b Rs2 Resolution between Imp-5 and Imp-6

c Rs3Resolution between Imp-6 and ZT

3754 Optimization of gradient program

Gradient mode of elution containing mobile phase-A (1mL of TEA1L

phosphate buffer pH adjusted to 55ACNMeOH 602020 vvv) and mobile

phase-B (1mL of TEA1L phosphate buffer pH adjusted to 55 ACN 4555

vv) were chosen to separate both the critical pairs in a single run and this

resulted in the co-elution of Imp-5 and Imp-6 peaks (Fig 35)

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

156 Chapter 3

Fig 35 Co elution of imp-5 and imp-6 at pH 55

Based on the experimental data it was predicted that increase in mobile

phase pH during the gradient elution could resolve the Imp-5 and Imp-6

without compromising the total run time Hence two more experiments were

designed with a mobile phase-B as combination of water ACN (4555 vv) for

experiment-1 and Phosphate Buffer (pH 73) ACN (4555 vv) for experiment-

2 Acetonitrile was selected as eluting solvent in the mobile phase-B to increase

the solvent strength thereby to elute strongly retained compounds (Imp-8)

faster The RS2 was less than 10 for experiment-1 and RS

2 was 15 for

experiment-2 The separation between Imp-5 amp Imp-6 was obtained at B value

60 but the elution time of Imp-8 was still at gt10min A gradient ramp (B from

60 to 70) between 50min and 82min was added to the program to reduce the

run time So Imp-8 was eluted at 8min To further improve the RS2 gradient

program was optimized with a linear increase of mobile phase-B with varying

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

157 Chapter 3

time points An optimum resolution was obtained (Fig 36) when the gradient

program of section 22 was used In addition to this the elution time of imp-8

was reduced to 6 min in the modified gradient method

Fig 36 Baseline separations of zolpidem and its impurities

Placebo interference study was verified by injecting sample solutions of

placebo and no interference of placebo with all the impurities ie Imp-1 to Imp-

8 and ZT analyte peak was found

Fig 37 Placebo chromatogram for zolpidem tablets

Imp-

1 - 1

051

Imp-

2 - 1

231

Imp-

3 - 2

139

Imp-

4 - 2

692

Imp-

5 - 3

720

Imp-

6 - 3

892

ZT -

414

7

Imp-

7 - 5

096

Imp-

8 - 5

981

AU

-0020

-0015

-0010

-0005

0000

0005

0010

0015

0020

0025

0030

0035

0040

0045

0050

0055

0060

Minutes

050 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

158 Chapter 3

Three different lots of mobile phases were prepared and the consistency in

the resolution was verified (Table 34) A system suitability check (RS1 RS

2 amp

RS3) was incorporated to ensure the adequate system performance during the

regular analysis and method validation

Table 34 Reproducibility in separation of ZT and its impurities

Compound Average

RT (Min) RRTa (n=6)c USP Resolutionb USP Tailing

factor

(n=6)c (n=6)c

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT) of

Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6)

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

159 Chapter 3

Fig 38 Peak purity plot for ZT in optimized conditions

Where PA = Purity angle and TH = Purity threshold

3755 The finalized RP-UPLC conditions for the estimation of assay and

related substances of ZT

Apparatus An UPLC with VWD and integrator

Column Acquity UPLC HSS T3 C-18 100 mm length

21 mm id 18 micron particle size

Flow Rate 03 ml min

Wave length 254 nm

Load 10microL

Column temperature 250C

Diluent Acetonitrile (ACN) Water (82)

Buffer Buffer solution was prepared by adding 1mL

of Triethyl amine (TEA) to 1000mL of 10 mM

NaH2PO4 H2O

Mobile phase-A Mixture of buffer (pH adjusted to 55 with

H3PO4) ACN and MeOH in the ratio of

602020 (vvv) Mobile phase-B A mixture of buffer (pH adjusted to 73 with

H3PO4) and ACN in the ratio of 4555 (vv)

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

160 Chapter 3

Table 35 Gradient program

Time Mobile phase-A Mobile phase -B

001 95 5

25 95 5

35 40 60

50 40 60

82 30 70

85 95 5

100 95 5

Table 36 Retention times and relative retention times wrto ZT

SNo ImpurityAnalyte

name

Retention time ~Relative

retention time

1 Imp-1 1019 024 2 Imp-2 1217 029 3 Imp-3 2203 052 4 Imp-4 2815 066 5 Imp-5 3879 091 6 Imp-6 4035 095 7 ZT 4244 100

8 Imp-7 5118 121 9 Imp-8 6037 142

3756 Degradation behavior

The purpose of this study is to establish the fact that the inherent chemical

stability of the molecule remains intact during its existence in the solid dosage

form along with other excipients So forced degradation studies should be

conducted on the drug to generate product related variants which in turn can

help to establish degradation pathways and thereby to develop and validate

suitable analytical procedure [22]

In this perspective a series of induced degradation experiments were

conducted at a concentration of 50 μgmL of ZT in active pharmaceutical

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

161 Chapter 3

ingredients (API) and tablets Degradation was induced by external physico-

chemical forces such as acid (5N HCl at 60oC) base (5N NaOH at 60oC)

oxidative (50 H2O2 at 60degC) UV-irradiation (at 254nm for 7 days) and thermal

treatment (105oC for 7 days)

The purity of the peaks obtained from the stressed samples was verified by

using the PDA detector The purity angle and purity threshold parameters were

evaluated for all the stressed samples to demonstrate the homogeneity of the

analyte peaks Assay of stressed samples was performed by comparison with

reference standards and the mass balance was calculated

37561 Degradation in Acidic solution

A single major degradation product Imp-2 (Zolpidem Acid) was formed when

ZT allowed to hydrolyze under acidic (5N HCl at 60oC for 24 hrs) conditions The

degradation pathway is based on the premise that acid or base catalyzed

reaction between water and tertiary amide gives its parent carboxylic acid and

an amine Here the other hydrolytic product was dimethylamine Using LC-MS

in positive ion ESI the degradation product was found to have an mz value of

281 at RRT 03 (Fig 39) which was confirmed by spiking analysis with Imp-2

and UV spectral match

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

162 Chapter 3

Fig 39 Mass spectrum and Typical UPLC chromatogram of ZT acid hydrolysis

Peak purity was evaluated for ZT peak and the resulting plot had shown that

the peak is pure as shown in Fig 310

Fig 310 Peak purity plot for ZT acid hydrolysis

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

163 Chapter 3

37562 Oxidative conditions

Zolpidem experienced a total degradation of 18 when the drug treated with

peroxide (50 H2O2 at 60degC for 2h) A major (gt10) unknown degradation

product was at RRT~127 Fig 311

Fig 311 Typical UPLC chromatogram of ZT oxidative degradation

Mass spectrum had shown the mz value 227 in positive ESI mode This

predominant degradation product with a molecular weight of 226 could be

zolpyridine as depicted in Fig 312

Fig 312 LC-MS data of ZT oxidative degradation

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

164 Chapter 3

The formation of zolpyridine could be due to the simultaneous cleavage of

tertiary amide and imidazopyridine ring Interestingly Imp-1 amp Imp-2 peaks

were also formed during the peroxide degradation which was confirmed by

spiking analysis followed by spectral match with the known standards Imp-1

was the secondary hydroxylated degradation product originated from Imp-2 In

addition a low level degradation product at RRT~11 with mz 324 indicating

the possible formation of N-oxide or hydroxylated compound of zolpidem (Mwt

323) (Fig 313)

Fig 313 LC-MS data of ZT oxidative degradation 11 RRT impurity

Peak purity was evaluated for the final stressed solution in peroxide and the

plot had been shown in Fig 314 The data had revealed that peak was

homogeneous and no other peaks were masked under ZT

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

165 Chapter 3

Fig 314 Peak purity plot for zolpidem Tartrate oxidative stress sample

37563 Base hydrolysis

Similar to acid hydrolysis single major degradation product Imp-2

(Zolpidem Acid) was formed when ZT was allowed to hydrolyze under basic (5N

NaOH at 60oC for 90 min) conditions (Fig 315)

Fig 315 ZT Base degradation chromatogram

But there was a difference in the rate at which hydrolysis occurred

Zolpidem underwent acid hydrolysis at a slower rate than the base hydrolysis

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

166 Chapter 3

LCMS data had confirmed the formation of Imp-2 in base hydrolysis (Fig 316)

Fig 316 LCMS data for base hydrolysis

Peak purity data had shown that peak was pure in base hydrolysis (Fig 317)

Fig 317 Peak purity plot in zolpidem Tartrate base hydrolysis

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

167 Chapter 3

37564 Water hydrolysis

The drug was not much susceptible to water hydrolysis however Imp-2 was

formed at a much lower rate of degradation (lt 02 after 24 hr at 60oC) (Fig

318)

Fig 318 Typical UPLC chromatogram of ZT water hydrolysis

Peak purity data had shown that the peak was pure in water hydrolysis

Fig 319 Peak purity plot for ZT Water hydrolysis

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

168 Chapter 3

37565 Heat and light conditions

Degradation was not observed when ZT was subjected to light and heat

conditions Purity angle was less than purity threshold in all the stressed

samples purity flag ldquoNordquo indicated the homogeneity of the peak The principal

peak mz value [M+H+] 308 (Mwt=307) supported the identity of ZT in all the

stressed conditions

The peak homogeneity test was performed for the ZT drug substance and drug

product by using DAD The data was shown below (Table 37)

Table 37 Purity Angle Purity Threshold and Purity Flag values

Stressed Condition Purity

Angle

Purity

Threshold

Purity

Flag

Normal 0278 0485 No

Acid degradation 0482 0669 No

Base degradation 1732 2687 No

Water hydrolysis 0301 1485 No

Oxidative degradation 1117 4584 No

Thermal degradation 0589 0678 No

Photolytic degradation 0405 0721 No

Relative rates of degradation were plotted in all the stress conditions as shown

in Fig 320

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

169 Chapter 3

Fig 320 Rate of degradation in various stress conditions

376 Mass balance study

The mass balance ( assay + sum of all compounds (impurities +

degradation products)) results were calculated for all of the stressed samples

and were found to be more than 988 (Table 38) The purity and assay of ZT

were unaffected by the presence of its impurities and degradation products

demonstrating the stability-indicating nature of the developed UPLC method

80

85

90

95

100

105

0 3 6 9 12 15 18 21 24

A

ssa

y

Time (in hours)

Rate of degradation in various stress conditions

Acid hydrolysis

Base hydrolysis

Peroxide degradation

Water hydrolysis

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

170 Chapter 3

Table 38 Summary of mass balance data in various stress conditions

Degradation

condition

Time RS by UPLC

degradation

Assay

(ww on

anhydrous

basis)

Mass balance

(assay+deg

Products)

Acid degradation 24 hrs 57 934 991

Base degradation 90 mins 83 909 992

Water hydrolysis 24 hrs 02 996 998

Oxidative

degradation

2 hrs 180 808 988

Thermal

degradation

7 days 01 996 997

Photolytic

degradation

7 days 01 996 998

Mass balance data close to 100 indicates good correlation between assay and

degradation products formed

The data shown above indicating the developed RP-UPLC method that was

found to be specific and selective for ZT analyte peak in the presence of its

impurities and degradation products

38 Analytical method validation

The developed method was completely validated as per ICH and USP [11-12]

381 System suitability test

A mixture of ZT standard (50microgmL) Imp-1 to Imp-8(0075microgmL) solution

was injected into the chromatographic system and good resolutions (Rs)

between all impurities and ZT peak was observed as shown in (Fig 321 to Fig

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

171 Chapter 3

323) The system suitability RS1 RS

2 and RS3 were found to be greater than 15

(Table 39)

Fig 321 Typical chromatogram of Blank

Fig 322 Typical chromatogram of test sample

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

172 Chapter 3

Fig 323 Chromatogram of test sample spiked with impurities

Assay standard solution (50microgmL) was also injected n=6 times and the RSD

for ZT peak area and tailing factor was recorded

Table 39 System suitability data

Compound Retention time (min)

RRT a Resolution b

(n=6) Tailing factor c

(n=6)

Imp-1 1019 024plusmn002 - 12plusmn010

Imp-2 1217 029plusmn002 22plusmn05 12plusmn010

Imp-3 2203 052plusmn002 95plusmn080 12plusmn010

Imp-4 2815 066plusmn002 50plusmn050 13plusmn009

Imp-5 3879 091plusmn002 81plusmn080 11plusmn010

Imp-6 4035 095plusmn002 18plusmn02 11plusmn005

ZT 4244 095plusmn002 22plusmn030 13plusmn005

Imp-7 5118 121plusmn002 58plusmn17 10 plusmn009

Imp-8 6037 142plusmn005 52plusmn10 11plusmn010

a Relative retention times (RRT) were calculated against the retention time (RT)

of Zolpidem tartrate

b Resolutions were calculated between two adjacent peaks

c Mean plusmn RSD (n=6) for ZT peak at assay concentration level

RSD for ZT peak area at assay concentration level was less than 08

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

173 Chapter 3

382 Precision

The precision study was carried out for assay using UPLC method by

analyzing six individual solutions of ZT test sample and standard solution The

RSD of six assay determinations was less than 04 (Table 310) The above

UPLC precision data had shown good precision and therefore the method was

reproducible

Table 310 Assay by UPLC precision results

Preparation Assay

1 999

2 991

3 991

4 993

5 999

6 990

Mean 993

Stdev 040

RSD 04

Precision was checked for related substances by injecting six different

preparations (n=6) of ZT (50microgmL) drug substance which was spiked with

impurities (Imp-1 to Imp-8) at 015 (0075microgmL) wrto the test

concentration The percentage RSD for peak area of Imp-1 to Imp-8 for six (n=6)

preparations was calculated (Table 311)

These results had confirmed that the developed UPLC method for related

substances was precise

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

174 Chapter 3

Table 311 Related substances by UPLC precision results

PreNo Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

1 17968 17995 12867 25650 16722 12189 10181 10713

2 17361 17514 11614 24793 16979 11797 9627 10428

3 17549 17559 12123 25358 16230 11379 10532 11164

4 17943 18096 12675 24985 16050 12183 9680 10199

5 17271 16847 12939 24626 16553 12212 9965 10379

6 17530 16754 12267 24573 16865 12760 9452 10553

Mean 17604 17461 12414 24998 16567 12087 9906 10573

Stdev 292 562 509 428 364 463 402 337

RSD 17 32 41 17 22 38 41 32

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

175 Chapter 3

Impurity precision was determined on 3 days and ANOVA was calculated for all

the impurities RSD was also calculated for inter-day precision and results

were tabulated below (Table 312)

Table 312 ANOVA results for precision

SNo Name F critical F calculated RSD

1 Imp-1 36823 14302 18

2 Imp-2 36823 21217 34

3 Imp-3 36823 10732 29

4 Imp-4 36823 23109 19

5 Imp-5 36823 27039 21

6 Imp-6 36823 24048 18

7 Imp-7 36823 03180 34

8 Imp-8 36823 09564 31

Inter day precision (RSD) for assay method was found to be 03

Intermediate precision study was carried out for ZT impurities by a different

analyst using a different instrument and a different column The RSD values

were calculated and tabulated as shown below Table 313

Table 313 Method ruggedness (System-2 Column-2 and Analyst-2)

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

RSD 36 28 27 09 16 39 62 34

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

176 Chapter 3

The result had shown the excellent precision of the method and was thus

proved to be rugged

Intermediate precision for ZT assay was performed on a different column

with a different instrument by a different analyst The results had shown

excellent precision as shown in Table 314

Table 314 Assay ruggedness (System-2 Column-2 and Analyst-2)

Preparation Assay

1 992

2 993

3 990

4 994

5 991

6 993

Mean 992

Stdev 015

RSD 015

383 Limit of detection (LOD) and Limit of quantification (LOQ)

LOD and LOQ were determined for impurities and ZT based on signal to

noise ratio A series of diluted solutions were injected to achieve SN ratio

about 100 for LOQ and 30 for LOD The final concentrations and SN ratios

were reported below (Table 315)

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

177 Chapter 3

Table 315 LOD and LOQ values of ZT and its impurities

SNo

Name of the

impurity LOQ in microgmL LOD in microgmL

10 Imp-1 000053 000016

20 Imp-2 000056 000017

30 Imp-3 000122 000037

40 Imp-4 000070 000021

50 Imp-5 000080 000024

60 Imp-6 000097 000029

70 Imp-7 000162 000049

80 Imp-8 000165 000050

90 ZT 000058 000017

3831 Precision at Limit of quantification

The precision at quantification level for related substances method was also

checked by analyzing six individual solution preparations of Imp-1 Imp-2 Imp-

3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 and ZT at quantification level The results

had shown consistent peak areas The method was found to be repeatable and

reproducible at limit of quantification level with RSD lower than 44 (Table

316)

Table 316 LOQ level precision results

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8 ZT

RSD 44 28 17 36 28 29 20 41 13

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

178 Chapter 3

3832 Accuracy at LOQ

LOQ recovery experiments were conducted to determine the accuracy of

developed method at limit of quantification LOQ blend solution LOQ recovery

solution and test sample solutions were prepared separately Each solution was

injected in triplicate (n=3) and the recovery was determined Excellent

recoveries were obtained for imp-1 to imp-8 (Fig 324) The mean recoveries

were shown in Table 317

Fig 324 Accuracy at LOQ chromatogram for ZT impurities

Table 317 Mean recovery data for ZT impurities

SNo Name of the impurity Mean Recovery

1 Imp-1 955

2 Imp-2 1012

3 Imp-3 1044

4 Imp-4 993

5 Imp-5 1028

6 Imp-6 1044

7 Imp-7 987

8 Imp-8 1030

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

179 Chapter 3

384 Linearity

3841 Linearity of assay method

The linearity test solutions of assay method were prepared from stock

standard solution Six solutions of concentrations ranging from 25 to 150

(25 to 75 microgmL) of ZT assay concentration (5microgmL) were prepared and each

solution was injected in triplicate The peak areas were recorded and the mean

peak area was calculated (Table 318) The calibration curve was plotted

between concentration and mean peak area

Table 318 Linearity data for ZT assay method

SNo Level of

concentration Area mean area

Inj-1 50 387491

Inj -2 387640

Inj -3 386495 387209

Inj -1 75 555238

Pre-2 558542

Inj -3 559897 557892

Inj -1 100 761747

Inj -2 757099

Inj -3 760110 759652

Inj -1 125 935749

Inj -2 933310

Inj -3 932900 933986

Inj -1 150 1121624

Inj -2 1112798

Inj -3 1103885 1112769

Correlation coefficient 09998

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

180 Chapter 3

Fig 325 Linearity plot for ZT assay method

3842 Linearity of related compounds method

Linearity for ZT and related compounds (Imp-1 to Imp-8) was determined by

analyzing the solutions of concentrations ranging from LOQ to 200 (LOQ

00075 001875 00375 005625 0075 009375 01125 013375 and 015

microgmL) of the specification Each solution was injected (n=6) times and the

mean peak area was considered for linearity assessment Regression analysis

was performed for concentration versus peak area to determine R2 value and

correlation coefficient (r) The data were also treated with ANOVA to calculate

the F-significance value The results were tabulated in Table 319

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

181 Chapter 3

Table 319 Linearity results for ZT and its impurities

SNo Name Corrleation(r) Co-efficient of

determination (R2) F-Significance

1 ZT 09996 09991 221x10-10

2 Imp-1 09998 09995 376x10-11

3 Imp-2 09999 09998 453 x10-12

4 Imp-3 09996 09992 179 x10-10

5 Imp-4 09996 09993 129 x10-10

6 Imp-5 09993 09985 953 x10-10

7 Imp-6 09990 09980 263 x10-09

8 Imp-7 09991 09982 193 x10-09

9 Imp-8 09996 09991 221 x10-10

The results shown above had shown excellent linearity of the analyte peaks

The much lower values of p lt 005 from ANOVA test indicated the significant

relationship between the concentration and the peak response

385 AccuracyRecovery

3851 Accuracy of the assay method

The recovery of the ZT assay method was determined by injecting five

individual sample preparations (n=5) at levels 50 100 and 150 of analyte

with concentration 50microgmL for drug substance and drug product Each

solution was injected in triplicate (n=3) and the mean peak area was considered

for the calculation of amount recovered The amount recovered for all the test

solutions was determined against ZT reference standard Percent recovery was

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

182 Chapter 3

calculated in terms of amount recovered versus amount added The mean

recoveries were shown below (Table 320)

Table 320 Accuracy results of ZT assay method

SNo levels () mean recovery (n=3)

(For drug substance)

() mean recovery (n=3)

(For drug product)

1 50 997 988

2 100 992 989

3 150 989 985

RSD at 100level for drug substance and drug product were 058 and 055

The data had shown very good recoveries at all three point concentration

(50 100 and 150) levels wherein the mean recovery lay between 989

and 997 for drug substance and 985 to 989 for drug product Thus the

developed UPLC method had shown good accuracy

3852 The accuracy of the related substance method

The recovery of impurities in related compounds method was studied at

50 100 and 150 of the specification level (015)

Solutions were prepared three times with impurities (Imp-1 to Imp-8) at the

level of 0075 015 and 0225 (ie wrt 50microgmL test concentration) and

each prepared solution was injected three times into the liquid chromatographic

system The mean recoveries of each impurity were calculated (Table 321)

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

183 Chapter 3

Table 321 Accuracy study data for ZT impurities

Name

of the impurity

(n=3) Mean recovery

50 level 100 level 150 level

Recovery plusmn RSD Recovery plusmn RSD Recovery plusmn RSD

Imp-1 9697plusmn12 9797plusmn40 9683plusmn13

Imp-2 9423plusmn07 9879plusmn01 9567plusmn08

Imp-3 9760plusmn060 10015plusmn83 9780plusmn07

Imp-4 9527plusmn05 9866plusmn16 9777plusmn07

Imp-5 10083plusmn03 10250plusmn35 10133plusmn09

Imp-6 10157plusmn08 10346plusmn17 10040plusmn09

Imp-7 9777plusmn06 9785plusmn43 9837plusmn09

Imp-8 10213plusmn08 10165plusmn49 10120plusmn10

The related substances by the UPLC method had shown consistent recoveries

at all three level concentrations

386 Solution stability

The solution stability for ZT in diluent medium for the assay method and

related substances by UPLC method was carried out by leaving the test

solutions of samples in tightly closed flasks (VF) on bench top at room

temperature for five days The sample solutions shown above were determined

for assay content and impurity content with an interval of 1 day The

percentage RSD for assay content of ZT during solution stability experiments

was not more than 05 (Table 322) Thus the sample solutions were found to

be stable up to 5 days

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

184 Chapter 3

Table 322 Solution stability study of ZT assay

SNo Time interval ZT Assay

1 Initial 998

2 Day1 997

3 Day2 998

4 Day3 999

5 Day4 992

6 Day5 993

RSD 029

No significant change was observed in the impurity content (Imp-1 to Imp-8)

of spiked solutions during the study period Hence the sample solutions were

considered to be stable up to 5 days

Table 323 Solution stability results for ZT impurities

SNo Impurity Initial Day1 Day2 Day3 Day4 Day5 RSD

1 Imp-1 019 019 020 019 019 018 33

2 Imp-2 019 021 021 020 021 020 40

3 Imp-3 018 019 018 017 018 018 35

4 Imp-4 016 015 015 016 015 016 35

5 Imp-5 018 018 018 019 018 017 35

6 Imp-6 018 019 018 020 020 018 52

7 Imp-7 016 015 015 014 015 015 42

8 Imp-8 020 021 022 020 021 020 40

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

185 Chapter 3

387 Robustness

Experiments were carried out with deliberately varied method parameters

and the system suitability was evaluated The results were tabulated below

(Table 324)

Table 324 System suitability-Robustness study

Parameters Conditions Resolution

RS1 a RS

2 b

Plate counts

Temperature

(plusmn 5ordmC)

32ordmC 18 23 12500

27ordmC 16 23 12932

22ordmC 18 22 13895

Different flow

(plusmn 10 )

033 ml 18 21 10298

030 ml 18 23 12932

027ml 19 24 13580

Different organic ratio

Acetonitrile

(plusmn 10)

110 17 23 9685

100 18 23 12932

90 17 23 14254

Buffer pH

(plusmn 01) in

mobile phase-A

54 17 23 9356

55 18 23 12932

56 18 24 14268

Buffer pH

(plusmn 01) in mobile phase-B

72 19 23 11874

73 18 23 12932

74 18 23 12864

aRS1 Resolution between imp-5 and imp-6

bRS2 Resolution between imp-6 and ZT

Tailing factor Tf was less than 12 in all the varied experimental conditions

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

186 Chapter 3

Table 325 Summary of ZT analytical method validation

Related substances by UPLC

Name Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-7 Imp-8

F critical 36823 36823 36823 36823 36823 36823 36823 36823

F calculated 14302 21217 10732 23109 27039 24048 003180 09564

p-value 02700 01543 03667 01333 00993 012419 07323 04064

Precision (RSD)

17 32 41 17 22 38 41 38

LOD(microgmL) 000016 000017 000037 000021 000024 000029 000049 000050

LOQ(microgmL) 000053 000056 000122 000070 000080 000097 000162 000165

Inter day

precision 18 34 29 19 21 18 34 31

Linearity Correlation(r) 09998 09999 09996 09996 09993 09990 09991 09996

ANOVA

p-value 376

x10-11 453

x10-12 179

x10-10 129

x10-10

953 x10-10

263 x10-09

193 x10-09

221 x10-10

R2 value 09995 09998 09992 09993 09985 09980 09982 09991

Mean

recovery 980 988 1002 987 1025 1035 979 1017

Solution stability

5 days 5 days 5 days 5 days 5 days 5 days 5 days 5 days

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

187 Chapter 3

39 Impurity profile Impurity profile studies were carried out for Zolpidem tartrate process

samples using the finalized method conditions The results were

tabulated below (Table 326)

Table 326 Impurity levels in different samples of ZT

Batch Imp-1 Imp-2 Imp-3 Imp-4 Imp-5 Imp-6 Imp-8 SMUI

1 002 005 002 004 006 001 004 003

2 001 006 002 002 004 001 003 002

3 002 005 002 004 006 001 003 003

4 002 007 004 004 004 002 004 003

5 002 006 002 006 005 001 004 005

6 002 004 004 004 005 ND 003 004

7 001 005 003 002 006 ND 004 002

8 001 005 005 003 007 ND 003 002

9 002 004 003 002 006 002 002 003

10 002 006 004 002 006 001 003 004

Mean 0017 0053 0031 0033 0055 00129 0033 0031

SD 00048 00095 0011 00134 00097 00049 00067 00099

Imp-7 was not detected in all the samples shown above

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

188 Chapter 3

Fig 326 Graphical representation of impurity profiles of ZT samples

310 Conclusion

A reversed phase-UPLC gradient method was developed for the determination

of ZT its related process impurities and degradation products This method can

be applicable for both ZT drug substance and drug product The method was

found to be selective precise accurate linear rugged and robust The total run

time was 100 minutes which enabled fast estimation of the drug substance

The developed method was proved to be stability-indicating This method can be

used for regular analysis of bulk and formulation samples from production area

and for the analysis of ZT stability samples

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

189 Chapter 3

References

[1]

Prescribing Informationrdquo sanofi-aventis 2007 Retrieved 2011-08-29

[2] Depoortere H Zivkovic B Lloyd KG Sanger DJ Perrault G Langer SZ

Bartholini G Zolpidem a novel nonbenzodiazepine hypnotic I

Neuropharmacological and behavioral effects J Pharmacol Exp Ther

237(2) 1986 649-58

[3] httpwwwaccessdatafdagovdrugsatfda_docslabel2007019908s02

2lblpdf Prescribing Information from official Gazette of Food and Drug

Administration

[4] Scatton B Claustre Y Dennis T Nishikawa T Zolpidem a novel

nonbenzodiazepine hypnotic II Effects on cerebellar cyclic GMP levels

and cerebral monoamines J Pharmacol Exp Ther 1986 237(2) 659-65

[5] Peter Kovacic Ratnasamy Somanathan Zolpidem a clinical hypnotic that

affects electronic transfer alters synaptic activity through potential GABA

receptors in the nervous system without significant free radical

generation Oxid Med Cell Longev 2009 2(1) 52ndash57

[6] Yasareni Sumalatha Padi Pratap Reddy Ranga Reddy Bollikonda

Satyanarayana Synthesis and spectral characterization of process-

related substances to the hypnotic agent zolpidem ARKIVOC (vii) 2009

143-149

190 Chapter 3

[7] Nirogi RV Kandikere VN Shrivasthava W Mudigonda K Quantification

of zolpidem tartrate in human plasma by high performance liquid

chromatography with fluorescence detection J Chromat B 2004 811 59ndash

63

[8] Laviana L Mangas C Mari FF Bayod M Determination and in process

control of zolpidem synthesis by HPLC J Pharm Biomed Anal 2004 36

925ndash928

[9] Ring PR Bostick JM Validation of a method for the determination of

zolpidem tartrate in human plasma using LC with fluorescence detection

J Pharm Biomed Anal 2000 22 495-504

[10] BA El Zeany AA Moustafa NF Farid Determination of zolpidem

hemitartrate by quantitative HPTLC and LC J Pharm Biomedical Anal

2003 33 393-401

[11] United States Pharmacopoeial Forum 2010 Volume 34(6) 1487

[12] European Pharmacopoeia 2008 volume 60 3256 ndash 3257

[13] Kelani KM Selective potentiometric determination of zolpidem

hemitartrate in tablets and biological fluids by using polymeric membrane

electrode J AOAC Int 2004 87 1309-1318

191 Chapter 3

[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of

Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4

[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods

for determination of zolpidem tartrate in tablet formulation J Pharm

Bioallied Sci 2010 2(4) 365ndash368

[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral

fluid by liquid chromatographyndashtandem mass spectrometry Journal of

Chromatography B 2004 811 59ndash63

[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of

zolpidem in urine and hair application to two drug facilitated sexual

assaults Forensic Science International 2004 143 157-161

[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic

Multiobjective Optimization Approach in Evaluation of Chromatographic

Behaviour of Zolpidem Tartrate and Its Degradation Products

Chromatographia 2011 74 197ndash208

[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology

2005

[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient

high-performance liquid chromatography theory and applications

Journal of Chromatography A 2004 1060 165-175

192 Chapter 3

[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change

of mobile phase pH during gradient reversed-phase chromatography with

222-trifluoroethanol-water as mobile phase and its effect on the

chromatographic hydrophobicity index determination Journal of

Chromatography A 2002 954 77ndash87

191 Chapter 3

[14] KS Patil YV Pore SB Bhise Spectrophotometric Estimation of

Zolpidem in Tablets J Pharm Sci amp Res 2010 2 1-4

[15] Rajiv Chomwal Amit Kumar Anju Goyal Spectrophotometric methods

for determination of zolpidem tartrate in tablet formulation J Pharm

Bioallied Sci 2010 2(4) 365ndash368

[16] Pascal Kintz Marion Villain Bertrand Ludes Testing for zolpidem in oral

fluid by liquid chromatographyndashtandem mass spectrometry Journal of

Chromatography B 2004 811 59ndash63

[17] M Villain M Chegraveze A Tracqui B Ludes P Kintz Windows of detection of

zolpidem in urine and hair application to two drug facilitated sexual

assaults Forensic Science International 2004 143 157-161

[18] Marija Malesevic Ljiljana Zivanovic Ana Protic Zarko Jovic

Multiobjective Optimization Approach in Evaluation of Chromatographic

Behaviour of Zolpidem Tartrate and Its Degradation Products

Chromatographia 2011 74 197ndash208

[19] ICH Q2 (R1) Validation of Analytical Procedures Text and Methodology

2005

[20] Roman Kaliszan Pawel Wiczling Michal J Markuszewski pH gradient

high-performance liquid chromatography theory and applications

Journal of Chromatography A 2004 1060 165-175

192 Chapter 3

[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change

of mobile phase pH during gradient reversed-phase chromatography with

222-trifluoroethanol-water as mobile phase and its effect on the

chromatographic hydrophobicity index determination Journal of

Chromatography A 2002 954 77ndash87

192 Chapter 3

[21] Sonia Espinosaa Elisabeth Boscha Mart Rose sa Klara Valkob Change

of mobile phase pH during gradient reversed-phase chromatography with

222-trifluoroethanol-water as mobile phase and its effect on the

chromatographic hydrophobicity index determination Journal of

Chromatography A 2002 954 77ndash87