CHAPTER-2 A VALIDATED STABILITY-NDICATING...

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CHAPTER-2

A VALIDATED STABILITY-NDICATING

ANALYTICAL METHOD FOR THE

DETERMINATION OF IMPURITIES IN

MONTELUKAST SODIUM

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2.1 Introduction of Montelukast sodium and survey of analytical

methods

Montelukast Sodium, the active ingredient in Singular*, is a

selective and orally active leukotriene receptor antagonist that inhibits

the cysteinyl leukotriene cys LT1 receptor [1]. Montelukast sodium is

described chemically as [[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-

Quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-

methyethyl)phenyl]propyl]sulfinyl]methyl]cyclopropane acetic acid,

mono-sodium salt (Fig: 2.1). Montelukast sodium is a hygroscopic,

optically active, white to off-white powder. It is freely soluble in

ethanol, methanol, water and practically in soluble in acetonitrile. The

empirical formula is C35H35ClNNaO3S. The molecular weight of

Montelukast Sodium is 608.18.

Fig: 2.1 Chemical structure of Montelukast Sodium

N

CH3

HO

H3C

Cl

S COO-Na+

[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-Quinolinyl)ethenyl]phenyl]-3-[2-(1-

hydroxy-1-methyethyl)phenyl]propyl]-sulfinyl]methyl]cyclopropane

acetic acid, mono-sodium salt.

Molecular Formula C35H35ClNNaO3S

Molecular Weight 608.18

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The different analytical techniques reported so far for the

determination of this drug and its metabolites in biological samples

include capillary electrophoresis [2] and spectrophotometry [3]. The

determination of Montelukast sodium in plasma by RP-LC [4-6] and in

solid dosage forms by RP-LC [7-8] was also reported. Forced

degradation or stress studies of drug substance and products play an

integral role in the development of pharmaceuticals [9]. The results of

degradation studies facilitate the stability-indicating method (SIM)

development. As on date, no stability-indicating HPLC method for the

quantitative determination of montelukast in montelukast sodium

bulk drug was reported in the literature. The current ICH guidelines

requires that the analysis of stability samples should be done by using

stability-indicating methods (SIAM’S) developed and validated after

stress testing on drug under variety of conditions, including acid, base

hydrolysis, oxidation, photolysis and thermal degradation [10].

Unfortunately, this route for the development of stability-

indicating related substances method was not found in most of the

stability-indicating methods reported in literature [11]. The target is to

develop a suitable stability-indicating HPLC related substances

method for montelukast sodium. In this chapter we describe a

stability-indicating LC method for the determination of montelukast

sodium and its potential and degradation impurities and also the

method validation.

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2.2. Development of a novel stability-indicating analytical

method for Montelukast Sodium

2.2.1 Materials

Reference standard of Montelukast and seven impurities

namely, Imp-A, Imp-B, Imp-C, Imp-D, Imp-E, Imp-F, Imp-G (Fig: 2.2

(a) to Fig: 2.2 (g)) were synthesized and characterized by use of LC-MS,

NMR and IR in Aurobindo Pharma Ltd., Hyderabad, India.

Montelukast sodium were provided by Chemical Research Division of

Aurobindo Pharma Ltd. All reagents used were of analytical reagent

grade unless stated otherwise. Milli Q water, HPLC-grade acetonitrile,

HPLC-grade orthophosphoric acid (OPA) were purchased from Merck

(Darmstadt, Germany).

2.2.2 Equipment

The LC system was equipped with quaternary gradient pumps

with autosampler and auto injector (Alliance Waters 2695, Milliford,

MA, USA) controlled with Empower software (Waters). Stability studies

were carried out in humidity chamber (Thermo lab Humidity chamber,

India) and photo stability studies were carried out in a photo stability

chamber (sanyo photo stability chamber. Leicestershire, U.K). Thermal

stability studies were performed in a dry air oven ( Merck Pharmaterh,

Hyd. India ). The LCMS analysis was performed on waters quatramino

TM API system equipped with triple quadrapole check. (Mass Lynax

4.1).

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Fig: 2.2 Chemical structures of impurities of Montelukast Sodium

N

CH3

HO

H3C

S COOHO

Cl

Molecular formula C35H36ClN04S


1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-hydroxy-1-methylethyl)phenyl]propyl]sulfinyl]methyl]

Cyclopropane aceticacid (Imp-A1&A2)

Fig: 2.2 (a)

N

CH3

HO

H3C

S COOHO

Cl

O



1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-

hydroxy-1-ethylethyl)phenyl]propyl]sulfonyl]methyl] Cyclopropane aceticacid (Imp-B)

Fig: 2.2 (b)

N

CH3

HO

H3C

Cl

OH

Molecular formula C29H28ClN02


2-(2-(3(S)-(3-((1E)-2-(7-Chloro-2-quinolinyl)ethenyl)phenyl)-3-hydroxypropyl)-phenyl)-2-propanol] (Imp-C)

Fig: 2.2 (c)

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N

CH3

O

Cl

S COOH



1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-(2-

acetylphenyl)-propyl]thio]methyl] cyclopropane aceticacid [Imp-D]

Fig: 2.2 (d)

N

CH3

HO

H3C

Cl

S COOCH3



1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(1-

hydroxy-1-methylethyl)phenyl]-propyl]thio]methyl]

Cyclopropane aceticacid, methylester (Imp-E)

Fig: 2.2 (e)

N

CH3

H2C

Cl

S COOH



1-[[[(1R)-1-[3-[(1E)-2-(7-Chloro-2-quinolinyl)ethenyl]phenyl]-3-[2-(prop-1-en-2-yl)phenyl]propyl]thio]methyl] Cyclopropane aceticacid (Imp-F)

Fig: 2.2 (f)

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NCl

O

CH3

CH3

Molecular formula C29H26ClN0


7-Chloro-2- ((E)-2-(3-(1,3,4,5-tetrahydro-1,1-dimethylbenzo-[C]

oxepin-3-yl)phenyl)-vinyl) quinoline (Imp-G)

Fig: 2.2 (g)

2.2.3 Preparation of sample solutions

The stock solutions of montelukast sodium (1.04 mg/ml) and

spiked with 0.50% of Imp-A and 0.15% of Imp-B, Imp-C, Imp-D, Imp-

E, Imp-F and Imp-G with respect to the Montelukast analyte

concentration. The stock solutions were further diluted with diluent to

obtain a standard solution of 0.5 mg/ml (500 µg/ml) for related

substances determination. The specification limit of Imp-A 0.50% and

0.15% of Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G in

montelukast sodium bulk drug sample was 0.15% w/w.

2.2.4 Generation of stress samples

One lot of montelukast sodium drug substance selected for

stress testing. From the ICH stability guideline (Q1AR2): “Stress

testing likely to be carried out on a single batch of material [12].

Different kinds of stress conditions (i.e., acid hydrolysis, base

hydrolysis, oxidative stress, heat, humidity, and light) were employed

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on one lot of montelukast sodium drug substance based on the

guidance available from ICH stability guideline (Q1AR2).

The details of the stress conditions studies are as follows:

Stress study under hydrolytic condition:

a) Acid Degradation: drug in 1.0 M HCl solution was kept at 85°C

for 15 mins.

b) Base Degradation: drug in 5 M NaOH solution was kept at 85°C

for 120 mins.

c) Oxidative stress: drug in 3.0% H2O2 solution was kept at room

temperature for 10 mins.

d) Thermal stress: drug was subjected to dry heat at 80°C for 120

hrs.

e) Photolytic degradation: drug was subjected to UV at 254 nm (10

K Lux ) for 48 hrs.

The photolytic degradation studies was carried out by exposing

the Montelukast sodium samples in solid state to light producing on

overall illumination of not less than 1.2 million lux hours and an

integrated near ultraviolet energy of not less than 200 wh/m2, which

took about 10 days period in our photostability chamber.

2.2.5 Optimization of chromatographic conditions:

Forced degradation studies were performed to develop a

stability-indicating HPLC method for the quantitative determination

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and purity evaluation of Montelukast sodium bulk drug substance.

The main objective of the chromatographic method was to seperate

Montelukast from Imp-A (A1 and A2), Imp-B, Imp-C, Imp-D, Imp-E,

Imp-F and Imp-G. Impurities were coeluted using different stationary

phases such as C8, phenyl and cyano as well as different mobile

phases. During the evaluation of pH study, no effect was observed in

elution order and retention times towards acidic side. Elution of

impurities required higher ratios of organic modifier, hence 0.1% OPA

was chosen as buffer solution to rule out precipitation of aqueous salt

buffers with combination of higher organic modifier ratios. During the

evaluation of various column chemistries, C18 was observed to give

better resolution. Resolution between Montelukast and Imp-D was

critical and conditions were optimized. The chromatographic

separation was achieved on a Waters 250 x 4.6 mm, Atlantis dC18, 5

µm particles. The gradient LC method employs solution A and solution

B as mobile phase. The solution A contains aqueous 0.1%

orthophosphoric acid and solution B contains a mixture of water :

acetonitrile (5:95 v/v). The flow rate of the mobile phase was 1.5

ml/min and the peak shape of the Montelukast sodium was found to

be homogeneous and symmetrical. The HPLC gradient program was

set as: time% solution B: 0.01/60, 10/70, 15/90, 20/100, 30/100,

32/60, 40/60 with a post run time of 10 min. The column

temperature was thermostated at 20°C and the UV detection was

monitored at a wavelength of 225 nm. The injection volume was 20

µl. A mixture of water : methanol (30:70 v/v) was used as a diluent.

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In the optimized chromatographic conditions Montelukast, Imp-A (A1

and A2) , Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G were

separated with a resolution greater than 2, typical relative retention

times were approximately 0.40, 0.45, 0.55, 0.63, 1.04, 1.35, 1.45,

1.59 with respect to Montelukast eluted at 13.894. Degradation was

not observed in Montelukast sodium samples when subjected to

forced degradation studies like thermal, photolytic and base

hydrolysis. Montelukast sodium was degraded to Imp-A (11.6%) under

oxidation (3.0% H2O2/rt/10 mins, Imp-F (0.2%) under acidic

conditions (1.0M HCl/85°C/10 mins] and some unknown degradants

observed (10%) under photolytic conditions (10K Lux /48 Hours].

Peak purity test results done by using a PDA detector confirmed

that the Montelukast peak is homogenous and pure in all the

analyzed stress samples. The mass balance of Montelukast Sodium in

all stress samples was close to 99.5% (%Assay + %Degradation). It is

clearly indicating that the developed HPLC method was found to be

specific for Montelukast in presence of its all impurities (Imp-A (A1

and A2), Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G) and

degradation compounds.

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Optimized liquid chromatographic conditions

Column : Atlantis dC18, 250 x 4.6 mm, 5µ

particle size

Mobile phase : The solution A contains aqueous 0.1%

OPA and Solution B contains a mixture

of Water: Acetonitrile (5:95 v/v).

Pump mode : Gradient

Flow rate : 1.5 ml/min

Column oven temperature : 20°C

UV detection : 225 nm

Injection volume : 20 l

Run time : 30 min

Retention time : 13.894

Relative Retention Time (RRT) : Impurity-A (A1 and A2) about 0.40,

0.45,

Impurity-B about 0.55

Impurity-C about 0.63

Impurity-D about 1.04

Impurity-E about 1.35

Impurity-F about 1.45

Impurity-G about 1.59

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Diluent : A mixture of water : methanol

(30:70 v/v)

Figures:

Fig 2.3 to Fig 2.7 is the typical HPLC chromatograms showing

the degradation of Montelukast sodium in various stress conditions

and also the corresponding peak purity plots.

Fig: 2.3 Typical HPLC chromatograms of Acid hydrolysis

Fig: 2.3 (a)

Blank Chromatogram of Acid hydrolysis (1N HCl)

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Fig: 2.3 (b)

Fig: 2.3 (c) Peak purity plot of Acid hydrolysis

Purity Angle Purity Threshold Purity Flag Peak Purity

0.052

0.261 No Pass

Fig: 2.3 (c)

Montelukast Sodium stressed with 1N HCl at 85°C for 10 mins

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Fig: 2.4 Typical HPLC chromatograms of Base hydrolysis

Fig: 2.4 (a)

Fig: 2.4(b)

Montelukast Sodium stressed with 5N NaOH at 85°C for 120 mins

Blank Chromatogram of Base hydrolysis (5N NaOH)

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Fig: 2.4 (c) Peak purity plot of Base hydrolysis


0.015

0.267 No Pass

Fig: 2.4 (c)

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Fig: 2.5 Typical HPLC chromatograms of Peroxide degradation

Fig: 2.5 (a)

Fig: 2.5 (b)

Blank Chromatogram of Peroxide Degradation ( 3% H2O2)

Montelukast Sodium stressed with 3% H2O2 at room temperature (intial)

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Fig: 2.5 (c) Peak purity plot of Peroxide Degradation

Purity Angle

Purity Threshold Purity Flag Peak Purity

0.034

0.263 No Pass

Fig: 2.5 (c)

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Fig: 2.6 Typical HPLC chromatograms of Photolytic degradation

Fig: 2.6 (a)

Fig: 2.6 (b)

Blank

Montelukast Sodium stressed with 10K Lux for 48 hours

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Fig: 2.6 (c) Peak purity of Photolytic Degradation


0.048 0.307 No Pass

Fig: 2.6 (c)

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Fig: 2.7 Typical HPLC chromatograms of Thermal Degradation

Fig: 2.7 (a)

Fig: 2.7 (b)

Blank

Montelukast Sodium stressed at 80°C for 120 mins

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Fig: 2.7 (c) Peak purity plot of Thermal Degradation


0.026 0.311 No Pass

Fig: 2.7 (c)

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2.2.6 Validation of Analytical method and its results:

The developed and HPLC method was taken up to method

validation. The analytical method validation was carried out in

accordance with ICH guideline [13].

2.2.6.1 System suitability :

A mixture of Montelukast sodium reference standard, Imp-A,

Imp-B, Imp-C, Imp-D, Imp-E, Imp-F and Imp-G injections were

injected into HPLC system and good resolution was obtained between

impurities and Montelukast Sodium. The system suitability results

are tabulated (Table: 2.1). Typical Blank, Montelukast Sodium Sample

and SST Chromatograms (Fig: 2.8).

Table: 2.1 System Suitability results

Compound

(n=3)

No. of

theoretical

plates (N)

USP Tailing factor

(T)

USP Resolution

(Rs)

Imp-A1 12326 1.07 -

Imp-A2 13668 1.11 3.02

Imp-B 216870 1.15 6.02

Imp-C 17630 1.39 4.36

Montelukast 26040 1.07 2.86

Imp-D 60019 1.10 4.05

Imp-E 108491 0.97 4.91

Imp-F 1122525 1.17 2.81

Imp-G 120805 1.10 23.35

n = Number of determinations

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Fig: 2.8 Typical Blank, Montelukast Sodium Sample and SST Chromatograms

Fig: 2.8 (a)

Fig: 2.8 (b)

Blank

Montelukast Sodium Sample

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Fig: 2.8 (c)

2.2.6.2 Precision:

The precision of an analytical process expresses the closeness of

agreement (degree of scatter) between a series of measurements

obtained from multiple sampling of the same homogeneous sample

under prescribed conditions.

Precision may be considered at three levels: System precision,

Method precision and Intermediate Precision. Assay method precision

study was evaluated by carrying out six independent assays of

Montelukast Sodium test sample against qualified Montelukast

sodium reference standard and RSD of six consecutive assays was

0.6% (Table: 2.2 to Table: 2.4).

The results showed insignificant variation observed in response.

Which indicated that the assay method was repeatable with RSD’s

below 0.4%.

Montelukast Sodium sample spiked with impurities

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Table: 2.2 System Precision results of the Assay method

Injection ID Montelukast Area

1 1769.956

2 1769.788

3 1769.244

4 1769.040

5 1769.589

6 1768.719

Mean 1769.389

SD 0.472

% RSD 0.03

95% Confidence Interval

± 0.495

Table: 2.3 Method Precision results of the Assay method

Sample ID Assay (% w/w)

1 99.1

2 99.0

3 99.4

4 99.3

5 99.1

6 99.2

Mean 99.2

SD 0.15

% RSD 0.2

95% Confidence

Interval ± 0.2

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Table: 2.4 Intermediate Precision results of the Assay method

Sample ID Assay (% w/w)

1 99.0

2 99.3

3 99.4

4 99.5

5 99.2

6 99.0

Mean 99.2

SD 0.21

% RSD 0.2

95% Confidence

Interval ± 0.2

The precision of the related substance method was checked by

injecting six individual preparations of Montelukast (1.04 mg/ml)

spiked with 0.50% of Imp-A and 0.15% of Imp-B, Imp-C, Imp-D, Imp-

E, Imp-F and Imp-G with respect to the Montelukast analyte

concentration. The % RSD of the area percentage of each impurity

(imp-A, -B, -C, -D, -E, -F and -G) for six consecutive determinations

was respectively as below (Table: 2.5 to Table: 2.7 ].

The results showed insignificant variation in measured

response. Which demonstrated that the related substances method

was repeatable with RSD’s below 1.3%.

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Table: 2.5 System Precision results of the Related substance

method

Injection ID Montelukast Area

1 44290

2 44578

3 44307

4 44430

5 44579

6 44352

Mean 44423

SD 0.3

% RSD 0.29

95% Confidence

Interval ± 136

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Table: 2.6 Method Precision results of Related Substance method

Preparation Imp-A (Sum of Imp-A1&

Imp-A2

Imp-B Imp-C Imp-D

1 0.513 0.166 0.167 0.133

2 0.519 0.168 0.169 0.134

3 0.512 0.167 0.168 0.135

4 0.513 0.167 0.168 0.133

5 0.520 0.169 0.172 0.135

6 0.519 0.169 0.170 0.133

Mean 0.516 0.168 0.169 0.134

%RSD 0.8 0.6 1.2 0.7

SD 0.004 0.001 0.002 0.001

95%

Confidence

level

±0.004 ±0.001 ±0.002 ±0.001

Preparation Imp-E Imp-F Imp-G

1 0.151 0.159 0.229

2 0.148 0.161 0.232

3 0.147 0.159 0.231

4 0.150 0.159 0.232

5 0.149 0.163 0.234

6 0.151 0.161 0.233

Mean 0.149 0.160 0.232

%RSD 1.3 1.3 0.9

SD 0.002 0.002 0.002

95% Confidence

level ±0.002 ±0.002 ±0.002

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Table: 2.7 Intermediate Precision results of Related

Substance method

Preparation Imp-A (Sum of Imp-A1&

Imp-A2

Imp-B Imp-C Imp-D

1 0.523 0.164 0.161 0.153

2 0.524 0.165 0.162 0.152

3 0.522 0.165 0.159 0.152

4 0.523 0.165 0.161 0.153

5 0.524 0.165 0.160 0.150

6 0.526 0.165 0.162 0.151

Mean 0.524 0.165 0.161 0.152

%RSD 0.2 0.0 0.6 0.7

SD 0.001 0.000 0.001 0.001

95%

Confidence level

±0.001 ±0.000 ±0.001 ±0.001

Preparation Imp-E Imp-F Imp-G

1 0.163 0.147 0.179

2 0.160 0.150 0.177

3 0.161 0.150 0.176

4 0.161 0.147 0.179

5 0.162 0.148 0.177

6 0.162 0.149 0.179

Mean 0.162 0.149 0.178

%RSD 0.6 0.7 0.6

SD 0.001 0.001 0.001

95% Confidence

level ±0.001 ±0.001 ±0.001

2.2.6.3 Limit of Detection (LOD) and Limit of Quantification

(LOQ)

The detection limit of an individual analytical procedure is the

lowest amount of analyte is a sample, which can be detected but not

necessarily quantitated as an exact value (Table: 2.8).

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The quantitation limit (LOQ) of an analytical procedure is the

lowest amount of analyte in a sample, which can be quantitatively

determined with suitable precision and accuracy. The quantitative

limit is a parameter of quantitative assays for low levels of compounds

in sample matrices, and is used particularly for the determination of

impurities and/or degradation products (Table: 2.9).

Table: 2.8 LOD values of the Montelukast Sodium and its

impurities

Preparation Imp-A Imp-B Imp-C Montelukast

1 1656 1393 1407 2027

2 2104 1743 1357 1773

3 1878 1532 1649 1563

4 1579 1698 1953 1475

5 1492 1373 1415 1652

6 1957 1315 1446 1703

Mean 1778 1509 1538 1699

SD 218 179 227 192

%RSD 12.3 11.9 14.8 11.3

Con. (µg/mL) 0.049 0.033 0.033 0.034

Con. (%w/w) 0.005 0.003 0.003 0.003

Preparation Imp-D Imp-E Imp-F Imp-G

1 1210 1097 1112 1086

2 1342 1117 1227 1451

3 1057 1426 1170 1593

4 1125 1492 1212 1420

5 1401 1386 1545 1188

6 1046 1253 1217 1107

Mean 1197 1295 1247 1308

SD 149 165 152 209

%RSD 12.4 12.7 12.2 16.0

Con. (µg/mL) 0.033 0.033 0.034 0.034

Con. (%w/w) 0.003 0.003 0.003 0.003

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Table: 2.9 LOQ values of the Montelukast Sodium and its

impurities

Preparation Imp-A Imp-B Imp-C Montelukast

1 5138 4736 4192 4048

2 5512 4823 4100 4042

3 5545 4507 4254 3904

4 5221 4280 4081 4234

5 5330 4462 4231 3859

6 5393 4511 4419 4175

Mean 5357 4553 4213 4044

SD 146 197 123 146

%RSD 2.7 4.3 2.9 3.6

Con. (µg/mL) 0.150 0.101 0.101 0.103

Con. (%w/w) 0.014 0.010 0.010 0.010

Preparation Imp-D Imp-E Imp-F Imp-G

1 3794 3861 3955 4295

2 3818 3918 3908 4040

3 3879 3869 3878 4041

4 3801 3875 4060 4220

5 4064 3802 3740 4222

6 3819 3677 4194 4282

Mean 3863 3834 3956 4183

%RSD 103 85 157 115

SD 2.7 2.2 4.0 2.7

Con. (µg/mL) 0.100 0.100 0.102 0.104

Con. (%w/w) 0.010 0.010 0.010 0.010

2.2.6.4 Linearity:

The linearity of an analytical procedure is its ability to obtain

test results, which are directly proportional to the concentration of

analyte in the test sample. The linearity of the assay method was

developed by injecting test sample at 80%, 90%, 100%, 110% and

120% of Montelukast sodium assay concentration (i.e.100 µg/ml).

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Each solution injected twice (n=2) into HPLC and the average area at

each concentration calculated (Table: 2.10).

Calibration curve drawn by plotting average area on the Y-axis and

concentration on the X-axis (Fig: 2.9).

Table: 2.10 Linearity results of the Assay method

% Concentration

Average area

80 1440311

90 1628455

100 1807701

110 1992448

120 2171422

Slope 35167

Intercept -18709

% Y - Intercept -1

Residual Sum of

Squares 2742

Correlation Coefficient 0.99997

Linearity Plot (Concentration Vs Response)

Fig: 2.9 Linearity Plot for Assay method

80.00 90.00 100.00 110.00 120.00

Aver

age

Are

a

Conc.(µg/mL)

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Linearity of the Related Substance method:

Linearity experiment were carried by preparing the Montelukast

sodium sample solutions containing Imp- A, Imp- B, Imp- C, Imp- D,

Imp- E, Imp- F and Imp- G from LOQ to 150% (i.e. LOQ 25%, 50%,

150%) with respect to their specifications limit (0.15%). Calibration

curve was drawn by ploting average value of the impurities. (Imp- A,

Imp- B, Imp- C, Imp- D, Imp- E, Imp- F and Imp- G on the y-axis and

concentrations on the X-axis (Fig: 2.10 to Fig: 2.18).

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Linearity results of the Related Substance method

Table: 2.11 Linearity results of the Imp-A

Imp-A

Concentration (µg/mL)

Area Statistical Analysis

0.150 5357 Slope 38088

0.262 9689 Intercept 103

0.524 19861 Residual

Sum of

Squares

762

0.786 30483

1.048 39591

1.310 51488

Correlation

Coefficient

0.9999 2.620 99414

3.929 149299

5.239 200055 Response

factor* 1.01

6.549 250469

Linearity Plot (Concentration Vs Area)

Fig: 2.10 Linearity plot for Imp-A

4403

24403

44403

64403

84403

104403

124403

0.259 1.259 2.259 3.259 4.259 5.259 6.259 7.259

Are

a

Con. (µg/mL)

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Table: 2.12 Linearity results of the Imp-B

Imp-B

Concentration

(µg/mL) Area Statistical Analysis

0.101 4553 Slope 45376

0.157 6545 Intercept -549

0.235 9725

0.314 13515 Residual Sum of

Squares

418 0.392 16921

0.785 35558

1.177 53131 Correlation Coefficient

0.9999 1.570 70055

1.962 88828 Response

factor 0.88

2.355 106243


Fig: 2.11 Linearity plot for Imp-B

4553

17553

30553

43553

56553

69553

82553

95553

0.101 0.501 0.901 1.301 1.701 2.101

Are

a

Con. (µg/mL)

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Table: 2.13 Linearity results of the Imp-C

Imp-C

Concentration


0.101 4213 Slope 40076

0.156 6593 Intercept 265

0.235 9083


Squares

440 0.391 15516

0.782 32150

1.173 47904 Correlation Coefficient

0.9999 1.565 62660

1.956 78666 Response

factor 1.00

2.347 94106


Fig: 2.12 Linearity plot for Imp-C

4213

16213

28213

40213

52213

64213

76213

88213

0.101 0.501 0.901 1.301 1.701 2.101

Are

a

Con. (µg/mL)

81

144

Table: 2.14 Linearity results of the Montelukast sodium

Montelukast sodium

Concentration


0.103 4044 Slope 39974

0.275 11091

0.549 22071 Intercept 149

0.824 32619


Squares

598 1.373 54310

2.746 110424

4.120 165626

Correlation

Coefficient 0.9999 5.493 219177

6.866 275159

8.239 328915 Response

factor 1.00


Fig: 2.13 Linearity plot for Montelukast sodium

4044

54044

104044

154044

204044

254044

304044

0.103 1.603 3.103 4.603 6.103 7.603

Are

a

Con. (µg/mL)

82

145

Table: 2.15 Linearity results of the Imp-D

Imp-D

Concentration


0.100 3863 Slope 38873

0.159 6425 Intercept 68

0.239 9267

0.319 12258 Residual Sum

of Squares 215

0.398 15659

0.797 30853

Correlation

Coefficient 0.9999 1.195 46918

1.593 61991

1.991 77591 Response factor

1.03 2.390 92745


Fig: 2.14 Linearity plot for Imp-D

3863

18863

33863

48863

63863

78863

0.100 0.500 0.900 1.300 1.700 2.100

Are

a

Con. (µg/mL)

83

146

Table: 2.16 Linearity results of the Imp-E

Imp-E

Concentration


0.100 3834 Slope 43454

0.160 6267

0.240 9781 Intercept -941

0.320 12823

0.400 15691 Residual Sum of Squares

413 0.799 33287

1.199 51554 Correlation

Coefficient 0.9999

1.599 68369

1.999 86069 Response factor

0.92 2.398 103304


Fig: 2.15 Linearity plot for Imp-E

3834

15834

27834

39834

51834

63834

75834

87834

99834

0.100 0.450 0.800 1.150 1.500 1.850 2.200

Are

a

Conc(µg/ml)

84

147

Table: 2.17 Linearity results of the Imp-F

Imp-F

Concentration


0.102 3956 Slope 43665

0.159 6435

0.239 10071 Intercept -309

0.319 13655


376 0.797 34790


Coefficient 0.9999

1.594 68774

1.992 86508 Response factor 0.92

2.390 104051


Fig: 2.16 Linearity plot for Imp-F

3956

18956

33956

48956

63956

78956

93956

0.102 0.402 0.702 1.002 1.302 1.602 1.902 2.202

Are

a

Conc(µg/ml)

85

148

Table: 2.18 Linearity results of the Imp-G

Imp-G

Concentration


0.104 4183 Slope 38372

0.158 5554

0.237 8635 Intercept -305

0.315 11653


347 0.788 30454


Coefficient 0.9998

1.577 59810

1.971 75137 Response factor 1.04

2.365 90762


Fig: 2.17 Linearity plot for Imp-G

4183

18183

32183

46183

60183

74183

88183

0.104 0.554 1.004 1.454 1.904 2.354

Are

a

Conc(µg/ml)

86

149

2.2.6.6 Accuracy/Recovery

The accuracy of an analytical procedure expresses the closeness

of agreement between the value, which is accepted either as a

conventional true value or an accepted reference value and the value

found.

Accuracy of the assay method

Accuracy of the assay method was developed by injecting three

different preparations of test sample at 80%, 100%, 120% of analyte

concentration (i.e.100 µg/ml). Each solution was injected twice (n=2)

into HPLC and the mean peak area of Montelukast sodium peak was

calculated.

Assay (%w/w) of test solution was determined against three

injections (n=3) of qualified Montelukast sodium standard (Table:

2.19).

The method was showed consistent and high absolute

recoveries at all three concentration (80%, 100%, 120% ) levels with

mean absolute recovery ranging from 99.3 % to 99.5%. The obtained

absolute recoveries were normally distributed around the mean with

uniform RSD values. The method was found to be accurate with low%

bias (< 1.0).

87

32

150

Table: 2.19 Accuracy results of the Assay method

S.NO Concentration

(%)

Mean recovery

(%)(n=3) %RSD

1 80 99.4 0.1

2 100 98.4 0.1

3 120 99.1 0.2

Accuracy of the related substances method established at 50% 100%

and 150% of the impurities specification limit (0.15%).

Accuracy at 50% impurity specification level:

Test solution prepared in triplicate (n=3) with impurities (Imp-A,

B, C, D, E, F and G) at 0.25% (Imp-A] and 0.1% (Imp- B, C, D, E, F, G)

level w.r.s analyte concentration (i.e 1.04 mg/m l). Each solution was

injected thrice into HPLC system (Table: 2.20).

88

151

Table: 2.20 Accuracy at 50% specification level

S.NO Impurity name Mean

recovery(%) SD %RSD

1 Imp-A1&Imp-A2 99.5

0.23

0.2

2 Imp-B 101.7 0.75 0.7

3 Imp-C 99.1 0.75 0.8

4 Imp-D 100.0 1.30 1.3

5 Imp-E 99.6 1.50 1.5

6 Imp-F 100.4 1.50 1.5

7 Imp-G 102.1 0.69 0.7



B, C, D, E, F and G) at 0.5% (Imp-A] and 0.15% (Imp- B, C, D, E, F, G)

level w.r.s analyte concentration (i.e 1.04 mg/m l). Each solution was

injected thrice into HPLC (Table: 2.21).


S.NO Impurity name Mean

recovery(%) SD %RSD

1 Imp-A1&Imp-

A2

98.5

0.12

0.1

2 Imp-B 102.1 0.97 1.0

3 Imp-C 97.5 0.40 0.4

4 Imp-D 100.2 0.40 0.4

5 Imp-E 95.8 0.97 1.0

6 Imp-F 95.6 0.38 0.4

7 Imp-G 103.0 0.97 0.9

89

152



B, C, D, E, F and G) at 0.75% of (Imp-A] and 0.25% of (Imp- B, C, D,

E, F, G) level w.r.s analyte concentration (i.e 1.04 mg/ml). Each

solution was injected thrice into HPLC system (Table: 2.22).


S.NO Impurity

name Mean

recovery(%) SD %RSD

1 Imp-

A1&Imp-A2 100.8 0.30 0.3

2 Imp-B 102.8 0.67 0.7

3 Imp-C 98.7 0.81 0.8

4 Imp-D 99.9 0.23 0.2

5 Imp-E 97.2 0.23 0.2

6 Imp-F 98.1 0.23 0.2

7 Imp-G 102.1 0.00 0.0

The related substance method was showed consistent and high

accurate recoveries of all six impurities at all three different

concentrations (50, 100, 150%) levels in drug substance.

2.2.6.7 Solution state stability:

The solution state stability of Montelukast sodium in diluent in

the assay method was carried out by leaving both the test solutions of

sample and reference standard in tightly capped volumetric flasks

kept at room temperature for two days. The same sample solutions

were assayed for every one hour interval up to the study period. The

%RSD of assay of Montelukast during solution stability experiments

was with in 1.0%.

90

153

The solution state stability of Montelukast sodium related

substance method was carried out by leaving sample solution in

tightly capped volumetric flask at room temperature for two days.

Content of impurities (Imp A, B, C, D, E, F and G) were checked for

every six hours internal up to the study period. No significant change

was observed in the content of all six impurities in drug solution

stability experiments up to the study period. Hence Montelukast

sodium sample solutions are stable for atleast 48 hours in the

developed method.

In assay method the standard and test solution injected at each

0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h. (Table: 2.23).

91

154

Table: 2.23 Solution stability results of the Assay method

S.No Time in Hours Assay (% w/w)

1 initial 99.1

2 1 99.0

3 2 99.5

4 3 99.6

5 4 99.1

6 5 99.2

7 6 99.1

8 7 99.0

9 8 99.4

10 9 99.3

11 10 99.1

12 11 99.2

13 12 99.1

% RSD 0.42

In related substances method the stability of Montelukast

sodium sample in diluent was established for 15 hr by injecting test

solution for every one hour interval up to the study period. The

impurity profiles obtained at different interval were very consistent

and matched with initial value.

92

155

2.2.6.8 Robustness

The robustness of an analytical procedure is a measure of its

capacity to remain unaffected by small, but deliberate variations in

method parameters and provides an indication and of its reliability

during normal usage. To determine the robustness of the developed

analytical method experimental conditions were purposely altered and

the resolution between Montelukast and its Imp-D was evaluated. In

each of the deliberately altered chromatographic condition (flow rate

1.3 ml/min and 1.7ml/min, acetonitrile 58% and 62% in the mobile

phase, column temperature 15C and 25C) the resolution between

Imp-B, Imp-C and Imp-D, Imp-E and Imp-F was greater than 2.0, to

illustrating the robustness of the method.

2.2.6.9 Mass balance

The mass balance is a process of adding both the assay value

and the levels of degradation products to see how closely these add up

to 100% of the initial value, with due consideration of the margin of

analytical error. Its establishment hence is a regulatory requirement.

The mass balance is very closely linked to the development of

stability- indicating assay method as it acts as an approach to

establish its validity. The stressed studies samples of Montelukast

sodium bulk drug were assayed against the qualified reference

standard and the results of mass balance obtained were very close to

99.8%. The results of mass balance obtained in each condition is

presented below (Table: 2.24).

93

156

Table: 2.24 Mass balance of the Assay method

Degradation

Mechanism

Degradation

Condition

% Assay

of active substance

Mass balance

(% Assay+ %

impurities+ %

degradants)

Remarks

Acid

1M HCl /

85°C/15 min.

80 99.8

Degraded to Imp-

F and some

unknown degradants

observed

Base

5M NaOH /

85°C /120min

99.5 99.7 No degradation

observed

Peroxide 30% H2O2/

RT /10 min 87 99.6

Degraded to Imp-

A and some

unknown degradants

observed

Thermal 80°C/120

Hours 98 99.7

No degradation

observed

Photolytic 10K Lux/48

Hours 90 99.5

Some unknown degradants

observed

2.3 Analysis of Montelukast sodium drug substance stability

samples

One manufacturing lot of Montelukast drug substance was

placed on stability study in chambers maintained at ICH set

conditions [12]. The analysis of stability samples were carried up to 24

months period using the above optimized method. The stability data

results obtained are presented in Table: 2.25 and Table: 2.26. The

developed HPLC method performed satisfactorily for the quantitative

evaluation of stability samples.

94

157

Table: 2.25 Accelerated stability data ( Storage conditions

40°C/75%RH)

Batch No: PS(781)194 Packing & storage conditions: Each sample

packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum

Stability study duration: 6 months Temperature

%Relative humidity

40°C/75%RH

Tests Description

Water

content (%w/w,

KF)

Identification

Assay (By HPLC,

%w/w, on

anhydrous basis)

Specifications

A white to off-white,

amorphous

powder

NMT 0.5

IR spectrum

should

concordant with that of

standard

NLT 98.0 and

NMT 101.0

Initial A white

powder 0.42 Complies 99.7

1M A white


2M A white


3M A white powder

0.59 Complies 99.3

6M A white


Related substances details on next page.

95

158

Related

Substances

LOQ

(%w/w)

LOD

(%w/w)

Related Substances (By HPLC, %w/w)

INITIAL 1M 2M 3M 6M

Imp-A1 0.014 0.005

ND ND ND ND ND

Imp-A2 0.04 0.04 0.05 0.05 0.06

Imp-B 0.010 0.003 ND ND ND ND ND

Imp-C 0.010 0.003 0.02 0.02 0.02 0.02 0.02

Imp-D 0.010 0.003 0.05 0.06 0.06 0.09 0.09

Imp-E 0.010 0.003 ND ND Below

LOQ

Below

LOQ

Below

LOQ

Imp-F 0.010 0.003 0.14 0.14 0.14 0.14 0.15

Imp-G 0.010 0.003 0.01 Below

LOQ 0.01 0.01 0.02

Highest unknown

- - 0.06 0.06 0.06 0.07 0.06

Total

unknown - - 0.15 0.12 0.06 0.11 0.07

Total RS - - 0.47 0.44 0.40 0.49 0.47

ND: Not detected NA: Not available

96

159

Table: 2.26 Long-Term stability data ( Storage conditions

25°C/60%RH)

Batch No: PS(781)19 Packing & storage conditions: Each sample

packed in a polyethylene bag in a triple laminated bag and kept in a HDPE drum

Stability study duration: 12 months Temperature

%Relative humidity

25°C/60%RH

Tests Description

Water

content (%w/w,

KF)

Identification

Assay (By HPLC,

%w/w, on

anhydrous basis)

Specifications

A white to off-white,

amorphous

powder

NMT 0.5

IR spectrum

should

concordant with that of

standard

NLT 98.0 and

NMT 101.0

Initial A white


1M A white


2M A white


3M A white powder

0.59 Complies 100.2

6M A white


9M A white


12M A white


24M A white powder

0.68 Complies 99.9

Related substances details on next page.

97

160

Related Substances

LOQ (%w/w)

LOD (%w/w)

Related Substances (By HPLC, %w/w)

Initial 3M 6M 9M 12M 24M

Imp-A1

0.014

0.005

Below LOQ

ND ND ND ND ND

Imp-A2 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-B 0.010 0.003 0.06 0.04 0.04 0.05 0.05 0.05

Imp-C 0.010 0.003 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-D 0.010 0.003 Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Below LOQ

Imp-E 0.010 0.003 0.03 0.03 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-F 0.010 0.003 0.03 0.03 Below

LOQ

Below

LOQ

Below

LOQ

Below

LOQ

Imp-G 0.010 0.003 ND ND ND ND ND ND

Highest

unknown - - ND ND ND ND ND ND

Total

unknown - - NA NA NA NA NA NA

Total RS - - 0.09 0.07 0.06 0.05 0.05 0.05

ND: Not detected

NA: Not available

98

161

2.4 Summary and Conclusions

Validated stability-indicating HPLC method was developed for

Montelukast sodium after subjecting the samples to stress testing

under ICH recommendes conditions. The RPLC method developed for

quantitative and related substance determination of Montelukast

sodium is rapid precise, accurate, linear and selective. The method was

completely validated and showing the satisfactory data for all the

method validation parameters tested. The developed method was found

to be ‘specific’ to the drug, as the peaks of the degradation products

did not interfere with the degradation peak. Thus the proposed method

can be employed for assessing the stability of Montelukast sodium

bulk drug samples.

99

100

Table: 2.27 Summary of Analytical method validation data

Test Parameter

Related Substances method Assay

method

Imp-A Imp-B Imp-C Imp-D Imp-E Imp-F Imp-G

Precision (RSD)

0.8 0.6 1.2 0.7 1.3 1.3 0.9 0.2

LOD (µg/ml)

0.049 0.033 0.033 0.033 0.033 0.034 0.034 N/A

LOQ (µg/ml)

0.150 0.101 0.101 0.100 0.100 0.102 0.104

N/A

Linearity (corre coefficient)

0.9999 0.9999 0.9999 0.9999 0.9999 0.9999 0.9999

0.9999

Accuracy (%) 98.4-101.1 101.3-103.4 97.3-100.0 98.7-101.3 94.7-101.3 95.3-101.3 101.3-103.8 99.4

Robustness

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Resolution b/w

Montelukast Imp-D>2

Solution stability Stable up to

15hr Stable up to

15hr Stable up to

15hr Stable up to

15hr Stable up to

15hr Stable up to

15hr Stable up to

15hr Stable up to

15hr

Mobile phase stability

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

Stable up to 15hr

101

2.5 References:

1. Physicians Desk Reference 63rd Edition., 2009, pp 2114.

2. Shakalisava, Y.; Regan, F.; J. Sep Sci., 2008, 31, 1137.

3. Alsarra, I.; Khalil, N. Y.; Sultan, M.; AL-Ashban, R.; Belal, F.;

Pharmazie., 2005, 60, 823.

4. Radhakrishna, T.; Narasaraju, A.; Ramakrishna, M.;

Satyanarayana, A.; J. Pharm. Biomed. Anal., 2003, 31, 359.

5. Sripalakit, P.; Kongthong, B.; Saraphanchotiuritthaya, A.;

J.Chromatogr., B Analyt Technol Biomed Life Sci. 2008, 869,

38.

6. Ochiai, H.; Uchiyama, N.; Takano, T.; Hara, K.; Kamei, T.;

J. Chromatogr., B 1998, 713, 409.

7. Smith, G. A.; Rawls, C. M.; Kunka, R. L.; Pharm Res., 2004, 21,

539.

8. AL-Rawithi, S.; AL-Gazlan, S.; AL-Ahmadi, W.; Alshowaier, I. A.;

Yusuf, A.; Raines, D. A.; J. Chromatogr., B: Biomed Sci., 2001,

754, 527.

9. Reynolds, D. W.; Facchine, K. L.; Mullaney, J. F.; Alsante, K.

M.; Hatajik, T. D.; Motto, M. G.; February 2002, 48.

10. Stability Testing of New Drug Substances and Products

(Q1AR2). ICH

Harmonised Tripartite Guideline.

11. Bakshi M.; Singh S.; J.Pharm. Biomed. Anal. 2002, 28, 1011.

12. Steven, W. Baertschi Pharmaceutical Stress Testing Predicting

Drug Degradation.

102

13. Validation of Analytical Procedures: Methodology Q2B–ICH

Guidelines.

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