SYNTHESIS AND CHARACTERIZATION OF SOME...

Chapter-3

A validated stability-indicating normal phase LC method for Clopidogrel bisulfate and its impurities in bulk drug and pharmaceutical dosage form

___________________________________________

3.1 Introduction of Clopidogrel bisulfate and survey of analytical

methods

Clopidogrel bisulfate is a thienopyridine class inhibitor of P2Y12 ADP

platelet receptors. Chemically it is methyl (+)-(S)-α-(2-chlorophenyl)-6,7-

dihydrothieno[3,2-c]pyridine-5(4H)-acetate sulfate. Clopidogrel bisulfate

is a white to off-white powder. It is practically insoluble in water at

neutral pH but freely soluble at pH 1. It also dissolves freely in methanol,

dissolves sparingly in methylene chloride, and is practically insoluble in

ethyl ether. The empirical formula of Clopidogrel bisulfate is

C16H16ClNO2S•H2SO4. The molecular weight of Clopidogrel bisulfate is

419.9.

Fig: 3.1.F1: Chemical structure of Clopidogrel bisulfate

methyl (+)-(S)-α-(2-chlorophenyl)-6,7-dihydrothieno[3,2-c]pyridine-5(4H)-

acetate sulfate

Molecular formula: C16H16ClNO2S•H2SO4, Molecular weight : 419.9

Clopidogrel is a potent anti-platelet and anti-thrombotic drug. It is a

dihydro thieno pyridine derivative pro-drug which is inactive in vitro. In

vivo, it selectively and irreversibly inhibits the binding of adenosine

diphosphate (ADP) to its platelet receptors [1]. Although the majority of

the drug is hydrolyzed by esterase to an inactive carboxylic acid

metabolite, the full anti-aggregating activity of the drug is achieved by

biotransformation to 2-oxo-Clopidogrel by cytochrome P450-1A. This

intermediate metabolite is hydrolyzed and generates an active form

which reacts as thiol reagent with the ADP receptor on platelets thus

preventing the binding of ADP. Clopidogrel has an absolute S-

configuration at carbon 7; the corresponding R-enantiomer is totally

devoid from anti-aggregating activity [2]. Both the carboxylic acid

hydrolysis product and the R-enantiomer are stated by the United States

Pharmacopeia (USP) as impurities A and C in the monograph, besides

impurity B which is a racemic mixture of regioisomer of Clopidogrel

(Impurity B1 and B2) [3]. USP method uses a glycoprotein based

ovomucoid column. The resolution of impurity B1 and B2 from

Clopidogrel is going bad in less number of injections in the USP method

(Fig: 3.1.F2). Apart from the monograph listed impurities, two more

process related impurities D and E are included in this study.

Few other methods have been reported for the estimation of

Clopidogrel by spectrophotometric [4, 5], by LC [6, 7], by CZE [8] and by

TLC [9]. A bio-analytical method for the determination of Clopidogrel in

plasma using LC–MS/MS was reported [10]. Clopidogrel was determined

by LC in the presence of its impurities using a chiral column and its

impurities were determined at low levels [11]. The carboxylic acid

metabolite (impurity A) was determined in human plasma by GC–MS

[12], by LC with UV detection [13] and by LC–MS [14]. The major

objective of the present work is to develop a single method for the

separation of related substance of Clopidogrel bisulfate as well as the

enantiomeric impurities with a short run time in drug substance and

drug product. Further the use of carbohydrate based chiral HPLC

column ensures the column life unlike glycoprotein based columns. So

far to our knowledge there was no method reported using normal phase

chromatography.

Fig: 3.1.F2: Typical chromatogram from USP Method

Hence a sensitive reproducible simple isocratic stability indicating

normal phase HPLC method was developed for quantitative

determination Clopidogrel bisulfate and its five impurities in

pharmaceutical dosage forms. The developed method was validated as

per ICH guidelines.

3.2. Development of stability-indicating normal phase HPLC method

for Clopidogrel bisulfate and its impurities

3.2.1 Materials

Tablets and standards of Clopidogrel bisulfate and its five impurities

namely impurity A, impurity B (racemic mixture of B1 and B2), impurity

C, impurity D and impurity E were supplied by Dr. Reddy’s laboratories

limited, Hyderabad, India. The HPLC grade n-hexane, ethanol and

analytical grade diethyl amine were purchased from Merck, Darmstadt,

Germany. High purity water was prepared by using Millipore MilliQ Plus

water purification system (Millipore, Milford, MA, USA).

3.2.2 Equipment

The Waters HPLC system (Waters, Milford, USA) used consists of a

pump, auto sampler and a PDA detector. The output signal was

monitored and processed using empower-2 software. Cintex digital water

bath was used for hydrolysis studies. Photo stability studies were carried

out in a photo stability chamber (Sanyo, Leicestershire, UK). Thermal

stability studies were performed in a dry air oven (Cintex, Mumbai, India)

3.2.3. Chromatographic conditions

The method was developed using Chiralcel OJ-H (250 mm x 4.6 mm,

5 μm) column. n- Hexane, Ethanol and diethyl amine in 95:5:0.05 v/v/v

ratio was used as mobile phase. The flow rate of the mobile phase was

1.0 ml/min. The column temperature was maintained at 25 °C and the

wavelength was monitored at 240 nm. The injection volume was 10 µl.

Fig: 3.2.F1: Chemical structures of impurities of Clopidogrel

Impurity-A (USP Related compound A)

Molecular formula: C15H14Cl NO2S

Molecular weight : 307.80

Impurity-D

Molecular formula :C7H9NS


Impurity-B (USP Related compound B)

Molecular formula: C16H16Cl NO2S

Molecular weight: 321.82

Impurity-E

Molecular formula:

C15H16ClNO2S


Impurity-C (USP Related compound C)

Molecular formula : C16H16ClNO2S Molecular weight : 321.82

3.2.4 Sample preparation

Stock solution of Clopidogrel (2.0 mg mL-1) was prepared by dissolving

appropriate amount of Clopidogrel bisulfate in ethanol. Working

solutions of 1000 and 100 µg mL-1in mobile phase were prepared from

the above stock solution for the related substance determination and

assay determination, respectively. A stock solution of impurity (mixture

of impurities A, B, C, D and E) at 0.5 mg mL-1 was also prepared in

ethanol.

3.2.5 Preparation of Tablets Sample Solution:

Twenty Clopidogrel bisulfate 75 mg tablets were weighed, transferred

to a clean and dry mortar and ground into a fine powder. Tablet powder

equivalent to 200 mg drug was then transferred to a 100 ml volumetric

flask, 70 ml of ethanol was added, and the flask was attached to a rotary

shaker for 10 min to disperse the material completely. The mixture was

then sonicated for 10 min and diluted to volume with ethanol to give a

solution containing 2000 μg mL-1. This solution was centrifuged at

3,000 RPM for 5 min and supernatant was diluted using mobile phase to

give test solutions containing 1000 μg mL-1 and 100 μg/ml. These

solutions were filtered through a 0.45 μm pore size Nylon 66 membrane

filter

3.2.6 Generation of stress samples

One lot of Clopidogrel bisulfate tablets was selected for stress testing.

From the ICH Stability guideline: “stress testing is likely to be carried out

on a single batch of material. Different kinds of stress conditions (i.e.,

heat, humidity, acid, base, oxidative and light) were employed on one lot

of Clopidogrel bisulfate tablets based on the guidance available from ICH

Stability Guideline (Q1AR2). The details of the stress conditions applied

were as follows:

a) Acid hydrolysis: Drug solution in 0.5 N HCl was exposed at at 80°C

for 24 h. b) Base hydrolysis: Drug solution in 0.5 N NaOH was exposed

at 80°C for 24 h. c) Oxidative stress: Drug solution in 3% H2O2 v/v was

exposed at room temperature for 24 h. d) Water hydrolysis: Drug

solution in water was exposed at 80°C for 24 h. e) Thermal stress:

Tablets were subjected to dry heat at 60°C for 10 days. f) Photolytic

degradation: The photo degradation was carried out by exposing the

Clopidogrel bisulfate tablets in solid state to light providing an overall

illumination of not less than 1.2 million lux hours and an integrated near

ultraviolet energy of not less than 200 W h/m2, which took about 10

days period in photo stability chamber .

3.3 Method development and optimization of chromatographic conditions

Forced degradation studies were performed to develop a stability

indicating HPLC method for the quantitative determination and purity

evaluation of Clopidogrel bisulfate. Stressed samples obtained during

forced degradation studies including the samples of Impurity-A,

Impurity-B, Impurity-C, Impurity-D and Impurity-E were utilized in the

HPLC method development.

3.3.1 Selection of wavelength

Impurities and Clopidogrel bisulfate solutions were prepared in

diluent at a concentration of 100 µg/ml and scanned in UV-Visible

spectro photometer; both the impurities and Clopidogrel bisulfate was

having UV maxima at around 240 nm (Fig: 3.3.F1). Hence detection at

240 nm was selected for method development purpose.

Fig: 3.3.F1: Typical UV spectrums of Clopidogrel and its impurities

A

b

so

r

b

an

c

e

Impurity-A

Impurity-B

Clopidogrel

Impurity-D

3.3.2 Column Selection

The objective of the chromatographic separation was to separation

impurity-A, impurity-D and impurity-E along with separation of

impurity-B (impurity-B1), enatiomer of impurity-B (impurity-B2), and

enatiomer Clopidogrel (impurity-C) from Clopidogrel. Since the method

involves the separation of chiral and non chiral impurities, initially trials

were conducted using reverse phase chromatography using different C18

columns, but separation was poor for chiral impurities.

Initially trials were conducted using different chiral stationary phases

like carbohydrates (cellulose and amylose) based columns. Trials were

conducted using 250 x 4.6 mm, 5 µm CHIRALPAK®AD column, 250 x

4.6 mm, 5 µm CHIRALCEL® OD column and 250 x 4.6 mm, 5 µm CHIRALCEL® OJ-H columns. n-Hexane and Ethanol in the ratio 95:5

(v/v) was used as mobile phase at flow rate 1.0 ml/min. For

CHIRALPAK®AD (amylose based) column, the separation of impurities namely impurity-A, impurity-B (B1 and B2), impurity-C and impurity-D

was not adequate and impurity-E was co- eluted with Clopidogrel. For

CHIRALCEL® OD and CHIRALCEL® OJ-H (cellolose based) columns, the separation was satisfactory for impurities namely impurity-A, impurity-B

(B1 and B2), impurity-C and impurity-D, but impurity-E was eluted very

closely with Clopidogrel and retention of Clopidogrel was about 21 min. But separation of impurity-E from Clopidogrel was better in

CHIRALCEL® OJ-H column. Hence CHIRALCEL® OJ-H was chosen for

further method development.

Wavelength

(nm)

Impurity-C Impurity-E

Table 3.3.T1: Experimental results on different stationary phases

Column Parameter Imp-

A Imp-

D Imp-B1

Imp-C

Imp-B2

Imp-E

Clopidogrel

250 x 4.6mm,

CHIRALPAK®AD, 5 µm

Retention time

6.8 7.5 8.9 9.6 23.9 - 29.9

USP

Tailing 1.2 1.4 1.1 0.9 0.8 - 1.1

250 x 4.6mm,

CHIRALCEL® OD, 5 µm

Retention time

4.8 8.5 10.3 12.4 15.6 20.4 21.2

USP

Tailing 1.1 1.2 1.1 1.2 0.9 - 1.2

250x4.6mm,

CHIRALCEL® OJ-H

Retention time

4.9 8.7 10.8 12.9 16.3 21.2 22.3

USP

Tailing 1.3 1.2 1.1 1.2 0.9 1.2 1.3

CHIRALCEL® OD = Tris(3,5-dimethylphenylcarbamate) of cellulose

CHIRALPAK® AD= Tris(3,5-dimethylphenylcarbamate) of amylase

CHIRALCEL® OJ-H= tris(4-methyl benzoate) of cellulose

3.3.3 Effect of additive

To improve the resolution between the impurities, basic additives

triethyl ammine and diethyl amine (0.05%) were added to above selected

mobile phase (n-Hexane, Ethanol in 95:5 (v/v)). When diethyl amine was

used in mobile phase, the resolution between impurities was improved

when compared to addition of triethyl amine to the mobile phase. Hence

n- Hexane, Ethanol and diethyl amine in 90:10:0.05, v/v/v; was chosen

as mobile phase with 1.0 mL min-1 flow rate

3.3.4 Effect of Column Temperature

When the column temperature was at 25°C, the symmetry of

Clopidogrel peak was 1.3. When Temperature increased to 40°C, there

was no improvement in symmetry of Clopidogrel peak. Hence column

oven temperature was finalized as 25°C

3.3.5 Effect of Organic solvent

When isopropyl alcohol was used in place of ethanol (n- Hexane,

isopropyl alcohol and diethyl amine in the ratio 95:5:0.05 (v/v/v),

Clopidogrel peak was strongly retained (about 30 min) and also impurity-

E was eluted as a broad peak. When ethanol ratio was increased (polarity

of mobile phase was increased) to 10% from 5%, the resolution between

impurity-E and Clopidogrel was poor. Hence ethanol ratio in mobile

phase was optimized at 5% (n- Hexane, ethanol and diethyl amine in

95:5:0.05, v/v/v).

3.3.6 Optimized chromatographic conditions for the determination

of related substances and assay of Clopidogrel

Column : Chiralcel OJ-H (250 x 4.6) mm with 5 µm

particle size

Elution : Isocratic

Mobile phase : Hexane, Ethanol and diethyl amine in

95:5:0.05 (v/v/v) ratio

Flow rate : 1.0 ml/min

Column temperature : 25°C

Wavelength of detection : 240 nm

Injection volume : 10 l

Run time : 35 min

Diluent : Mobile phase

Retention time : Clopidogrel about 22 min

Relative Retention Time : Impurity-A - about 0.22

Impurity-B1 - about 0.50

Impurity-B2 - about 0.75

Impurity-C - about 0.59

Impurity-D- about 0.40

Impurity-E- about 0.90

The interference from excipients Mannitol, Colloidal Silicon Dioxide, Croscarmellose Sodium, Hydroxy Propyl Cellulose, Hydroxy Propyl

Methyl Cellulose, Hydrogenated Castor Oil Powder, microcrystalline

cellulose, Opadry Pink was also checked by injecting sample solutions of

excipients. There was no interference from excipients found at retention times of impurities (impurity-A, impurity-B1& B2, impurity-C, impurity-

D and impurity-E) and Clopidogrel peak.

3.4 Degradation behavior

HPLC studies on Clopidogrel bisulfate under different stress

conditions suggested the following degradation behavior.

3.4.1 Degradation in acidic solution

Clopidogrel bisulfate tablets powder in solution was exposed to 0.5 N

HCl at 80°C for 24 h, degradation was observed leading to the formation

Impurity-A (Fig: 3.4.F1 (a) to Fig: 3.4.F1 (b)).

Fig: 3.4.F1 (a): Typical HPLC chromatogram of Acid hydrolysis

Fig: 3.4.F1 (b): Peak purity plot of Clopidogrel under Acid hydrolysis

Purity Angle Purity

Threshold Purity Flag Peak Purity

0.274 0.353 No Pass

3.4.2 Degradation in Basic solution

Clopidogrel bisulfate tablets powder in solution was exposed to 0.5 N

NaOH at 80°C for 24 h, degradation was observed leading to the formation Impurity-A (Fig: 3.4.F2 (a) to Fig: 3.4.F2 (b)).

Fig: 3.4.F2 (a): Typical HPLC chromatogram of alkali hydrolysis

Fig: 3.4.F2 (b): Peak purity plot of Clopidogrel under alkali

hydrolysis

Purity Angle Purity

Threshold Purity Flag Peak Purity

0.341 0.355 No Pass

3.4.3 Oxidative conditions

Clopidogrel bisulfate tablets powder in solution was exposed to 3% hydrogen peroxide at room temperature for 24 h. The drug gradually

undergone degradation with time in 3 % hydrogen peroxide and mild

degradation was observed (0.6%) (Fig: 3.4.F3 (a) to Fig: 3.4.F3 (b)).

Fig: 3.4.F3 (a): Typical HPLC chromatogram of oxidative degradation

Fig: 4.4.F3 (b): Peak purity plot of Clopidogrel under oxidative

degradation

Purity Angle Purity

Threshold Purity Flag Peak purity

0.133 1.442 No Pass

3.4.4 Degradation in Neutral (Water) solution

No major degradation products were observed after 24 h at room

temperature. The drug was also stable in water on heating at 80°C for

24h. The drug was stable to water hydrolysis (Fig: 3.4.F4 (a) to Fig: 3.4.F4 (b)).

Fig: 3.4.F4 (a): Typical HPLC chromatogram of Water hydrolysis

Fig: 3.4.F4 (b): Peak purity plot of Clopidogrel under Water

hydrolysis

Purity Angle Purity


0.513 0.591 No Pass

3.4.5 Photolytic conditions

When the drug powder was exposed to light for an overall illumination of 1.2 million lux hours and an integrated near ultraviolet

energy of 200-watt hours/square meter (w/mhr) (in photo stability

chamber), no degradation of the drug was observed. The drug was stable to photolysis (Fig: 3.4.F5 (a) to Fig: 3.4.F5 (b)).

Fig: 3.4.F5 (a): Typical HPLC chromatogram of Photo degradation

Fig: 3.4.F5 (b): Peak purity plot of Clopidogrel under Photo

degradation

Purity Angle Purity


0.460 0.597 No Pass

3.4.6 Thermal Degradation:

The drug was stable to the effect of temperature. When the drug

powder exposed to dry heat at 60°C for 10 days, mild decomposition of the drug was observed (Fig: 3.4.F6 (a) to Fig: 3.4.F6 (b)).

Fig: 3.4.F6 (a): Typical HPLC chromatogram of thermal degradation

Fig: 3.4.F6 (b): Peak purity plot of Clopidogrel under Thermal

degradation

Peak purity test performed for the Clopidogrel peak using photodiode

array (PDA) detector data confirmed the peak purity of the peak for all

the forced degradation samples. % Assay of all stressed samples were

calculated using qualified standard of Clopidogrel. Considering the

purities from the respective chromatograms of forced degradation

samples, mass balance (%assay + % degradents + % impurities) was

calculated for all stress samples. The mass balance of stressed samples

was close to 99%. This clearly demonstrates that the developed normal

phase HPLC method was specific for Clopidogrel in presence of its

impurities (Impurity-A, Impurity-B, Impurity-C, Impurity-D and

Impurity-E) and degradation products.

3.5 Analytical method Validation and its results

Purity Angle Purity

Threshold Purity Flag Peal purity

0.038 1.123 No Pass

The developed and optimized HPLC method was taken up for method

validation. The analytical method validation was carried out in

accordance with ICH guidelines [15, 16].

3.5.1 System Suitability Test

Clopidogrel bisulfate standard, impurity-A, impurity-B, impurity-C,

Impurity-D and impurity-E were spiked at 0.20% level with respect to the

concentration of Clopidogrel in diluent and injected for five times into

HPLC system. Resolution between impurities and Clopidogrel, tailing

factor, theoretical plates for impurities and Clopidogrel, RSD% for the

areas of impurities and Clopidogrel was calculated. Good resolution was

obtained between impurities and Clopidogrel (Fig: 3.5.F1). System

suitability results were tabulated (Table 3.5.T1).

Fig: 3.5.F1: Typical chromatogram of system suitability

Table 3.5.T1: System Suitability results

Compound

(n=5)

Resolution (Rs) USP

Tailing factor (T)

% RSD for

peak area

Impurity-A

- 1.2 0.5

Impurity-D

11.8 1.2 0.2

Impurity-B1

4.6 1.2 0.9

Impurity-C

4.3 1.2 0.5

Impurity-B2

6.0 1.1 0.3

Impurity-E

5.0 1.0 0.5

Clopidogrel

2.4 1.3 0.4

n = Number of determinations

3.5.2 Precision

Assay method precision (repeatability) study was evaluated by carrying

out six independent assays of Clopidogrel bisulfate test sample against

qualified reference standard and %RSD of six consecutive assays was

0.81% (Table 3.5.T2). Results showed insignificant variation in measured

response which demonstrated that the method was repeatable with RSDs

below 0.9%.

Table 3.5.T2: Precision results of the assay method

Preparation %Assay

1 99.2

2 100.5

3 99.9

4 99.4

5 100

6 98.2

Mean 99.5

SD 0.80

% RSD 0.81

95% Confidence interval of

mean 98.9 to 100.1

The precision of the related substance method was evaluated by

injecting six individual preparations of Clopidogrel (1.0 mg mL-1) spiked

with 0.20% of Impurity-A, Impurity-B, Impurity-C, Impurity-D and

impurity-E with respect to Clopidogrel analyte concentration. The % RSD for area of Impurity-A, Impurity-B, Impurity-C, Impurity-D and impurity-

E for six consecutive determinations was below 3.0% (Table 3.5.T3).

Results showed insignificant variation in measured response which demonstrated that the related substances method was repeatable with

RSDs below 3.0%.

Table 3.5.T3: Precision results of the RS method

Prepa

ration Impurity-A Impurity-B* Impurity-C Impurity-D Impurity-E

1 225053 48870 260280 188212 128917

2 221536 51509 266775 186777 129955

3 219594 48852 264130 187569 131354

4 218822 49369 269611 181313 126631

5 214859 49500 267868 188662 130236

6 218808 50550 258616 185682 134204

Mean 219778.6 49775 264546.7 186369.2 130216

SD 3376.4 1050.9 4363.3 2694.2 2522.1

%RSD 1.53 2.11 1.64 1.44 1.93

* Impurity B1+ B2

Intermediate precision for assay and related substances method was

performed by carrying out six independent assays of Clopidogrel bisulfate

tablets test sample against qualified reference standard and RSD of six

consecutive assays was calculated. Related substances method was

performed by injecting six individual preparations of Clopidogrel (1.0 mg

mL-1) spiked with 0.20% of Impurity-A, Impurity-B, Impurity-C,

Impurity-D and impurity-E with respect to Clopidogrel analyte

concentration over different days, different instruments and with

different analysts (Table 3.5.T4).

Table 3.5.T4: Results of Intermediate precision

S.No

Parameter

Variation

%RSD

for Assay

%RSD

for Related

Substances

1

Repeatability

(a) Analyst-1

(b) Waters 2695 Alliance system.

(c) Day-1

0.81%

< 3.0%

2

Intermediate

precision

(a) Analyst-2

(b) Agilent 1100 series

VWD system.

(c) Day-2

0.75%

< 3.0%

3.5.3 Limit of quantification (LOQ) and limit of detection (LOD)

LOQ and LOD were established for Impurity-A, Impurity-B, Impurity-

C, Impurity-D and impurity-E based on signal to noise ratio method.

3.5.3.1 Limit of quantification (LOQ)

LOQ was established for Clopidogrel and for impurity-A, impurity-B,

impurity-C, impurity-D and impurity-E based on signal to noise ratio method (Table 3.5.T5).

Table 3.5.T5: LOQ values of Clopidogrel and its impurities

S.No Impurity

name Concentration

Signal to noise ratio

1 Impurity-A 0.410 µg mL-1 11.2

2 Impurity-B 0.320 µg mL-1 9.1

3 Impurity-C 0.310µg mL-1 9.5

4 Impurity-D 0.360 µg mL-1 10.8

5 Impurity-E 0.360 µg mL-1 9.8

3.5.3.2 Limit of detection (LOD) The detection limits were established by S/N methodology and

established concentration values are reported in Table 3.5.T6.

Table 3.5.T6: LOD values of Clopidogrel and its impurities

S.No Impurity name Concentration Signal to noise

ratio

1 Impurity-A 0.160 µg mL-1 3.3

2 Impurity-B 0.140 µg mL-1 2.8

3 Impurity-C 0. 090µg mL-1 2.6

4 Impurity-D 0.110 µg mL-1 3.1

Impurity-E 0.130 µg mL-1 2.9

3.5.4 Precision at Limit of quantification

The precision of the related substance method was also checked by injecting six individual preparations of Impurity-A, Impurity-B, Impurity-

C, Impurity-D and impurity-E at their LOQ level with respect to

Clopidogrel analyte concentration. Results showed insignificant variation

in measured responses which demonstrate that the method was

repeatable at LOQ level with RSD below 4.0% (Table 3.5.T7).

Table 3.5.T7: Precision results of the RS method at LOQ level.

Preparati

on

Impurity-

A

Impurity-

B

Impurity-

C

Impurity-

D

Impurity-

E

1 29536 15961 20010 37004 27932

2 29761 15652 19604 37431 26795

3 30287 15342 20910 36660 28018

4 29974 15857 19835 36287 27583

5 29174 14407 20859 36627 27367

6 29959 15088 20322 37382 27814

Average 29782 15385 20257 36899 27585

Std Dev 388.29 578.34 540.10 454.53 454.31

%RSD 1.3 3.7 2.6 1.2 1.6

3.5.5 Accuracy at LOQ level

The recovery studies for Impurity-A, Impurity-B, Impurity-C, Impurity-D and impurity-E were carried out in triplicate at LOQ level of

the Clopidogrel target analyte concentration (1000 µg mL-1). The

percentage recovery of Impurity-A, Impurity-B, Impurity-C, Impurity-D and impurity-E was calculated (Table 3.5.T8). The method showed

consistent and high absolute recoveries at LOQ level with mean absolute

recovery ranging from 98.1 % to 99.3%. The obtained absolute recoveries were normally distributed around the mean with uniform RSD values.

The method was found to be accurate with low % bias (< 2.0).

Table 3.5.T8: Recovery at LOQ level

S.No

Impurity name

Mean recovery (%)

(n = 3 )

%RSD

1 Impurity-A 98.1 0.31

2 Impurity-B 98.5 0.22

3 Impurity-C 98.9 0.33

4 Impurity-D 99.1 0.35

5 Impurity-E 99.3 0.18

3.5.6 Linearity

3.5.6.1 Linearity of the assay method

The linearity of the assay method was established by injecting test

sample at 50%, 75%, 100%, 125% and 150% of Clopidogrel bisulfate

assay concentration (i.e.100 µg mL-1). Each solution was injected in to

HPLC system (Table 3.5.T9). Calibration curve obtained by analysis

between peak area and the concentration showed (Fig: 3.5.F2) linear

relationship with a regression coefficient 0.9999. The best fit linear

equation obtained was y = 10651 x con + 93892.

Analysis of residuals indicated that the residuals were normally (Table

3.5.T10) distributed around the mean with uniform variance across all

concentrations suggesting the homoscedastic nature of data.

Table 3.5.T9: Linearity results of the assay method

Concentration (%) Peak area

50 5433303

75 8044168

100 10819223

125 13497932

150 16050284

Correlation

Coefficient(r) 0.99991

Slope 106751

Intercept 93892

Standard error 644278.0

Fig: 3.5.F2: Linearity plot for Assay Method

Table 3.5.T10: Residual summary of Linearity results of the assay

method

%conc

Mean area

Response achieved

Response calculated

thru Trend

line equation

Residual

(Response

practical -Response

theoretical)

Residual

square

50 5433303 5431436.80 -1866 3482702.4

75 8044168 8100209.40 56041 3140638514.0

100 10819223 10768982.00 -50241 2524158081.0

125 13497932 13437754.60 -60177 3621319470.8

150 16050284 16106527.20 56243 3163297546.2

Residual sum of squares

12452896314.4

Trend line equation y = 10675x + 93892

3.5.6.2 Linearity of the related substances method

Linearity experiments were carried out by preparing the Clopidogrel

bisulfate sample solutions containing Impurity-A, Impurity-B, Impurity-C, Impurity-D and impurity-E from LOQ to 150% (i.e. LOQ, , 50%, 75%,

100% and 150%) with respect to specification limit 0.20% (Table

3.5.T11).

Calibration curve was drawn by plotting peak area of the impurity on

the Y-axis and concentration on the X-axis (Fig: 3.5.F3 to Fig: 3.5.F7)

which showed linear relation ship with a regression coefficient of greater

than 0.998 for all impurities. Analysis of residuals showed (Table 3.5.T12 to Table 3.5.T16) that the residuals were normally distributed around the

mean with uniform variance for all concentrations.

Table 3.5.T11: Linearity results of the RS method

S.No

% of impurity

w.r.t

1000 µg/ml

Impurity-A (Peak area)

Impurity-B (Peak area)

Impurity-

C (Peak

area)

Impurity-

D (Peak

area)

Impurity-E (Peak area)

1 LOQ 25477 15772 18786 37490 27451

2 0.10 64056 29005 46188 85817 65263

3 0.15 93814 43700 75471 134565 103809

4 0.20 134091 57814 100288 177444 135244

6 0.30 198079 85053 148587 264424 200325

Correlation

coefficient (r) 0.9992 0.9999 0.9991 0.9995 0.9989

intercept -7213 1694 -5254 -4910 -3773

Slope 689479 278588 519493 905366 688696

Fig: 3.5.F3: Linearity plot for impurity-A

Fig: 3.5.F4: Linearity plot for impurity-B

Fig: 3.5.F5: Linearity plot for impurity-C

Fig: 3.5.F6: Linearity plot for impurity-D

Fig: 3.5.F7: Linearity plot for impurity-E

Table 3.5.T12: Residual summary of Linearity results of impurity-A

% of

impurity

w.r.t 1000

µg/ml

Mean area

Response achieved

Response

calculated

thru Trend line

equation

Residual

(Response

practical -Response

theoretical)

Residual

square

0.05 25477 27260.69 1783.7 3181547.1

0.10 64056 61734.65 -2321.4 5388672.1

0.15 93814 96208.61 2394.6 5734148.0

0.20 134091 130682.57 -3408.4 11617411.6

0.30 198079 199630.49 1551.5 2407110.3

Residual sum of squares 28328889.2

Trend line equation y = 689479.189x - 7213.270

Table 3.5.T13: Residual summary of Linearity results of impurity-B

% of

impurity w.r.t

1000

µg/ml

Mean area Response

achieved

Response

calculated thru Trend

line

equation

Residual

(Response practical -

Response

theoretical)

Residual

square

0.05 15772 15624.11 -147.9 21872.0

0.10 29005 29553.51 548.5 300867.1

0.15 43700 43482.92 -217.1 47124.2

0.20 57814 57412.32 -401.7 161343.3

0.30 85053 85271.14 218.1 47582.9


578789.6

Trend line equation y = 278588.108x + 1694.703

Table 3.5.T14: Residual summary of Linearity results of impurity-C

% of

impurity

w.r.t 1000

µg/ml

Mean area

Response achieved

Response

calculated

thru Trend line

equation

Residual

(Response

practical -Response

theoretical)

Residual

square

0.05 18786 20719.74 1933.7 3739362.9

0.10 46188 46694.41 506.4 256446.4

0.15 75471 72669.07 -2801.9 7850825.4

0.20 100288 98643.73 -1644.3 2703624.7

0.30 148587 150593.05 2006.1 4024252.9

Residual sum of squares 18574512.3


Table 3.5.T15: Residual summary of Linearity results of impurity-D

% of

impurity

w.r.t 1000

µg/ml

Mean area

Response achieved

Response

calculated

thru Trend line

equation

Residual

(Response

practical -Response

theoretical)

Residual

square

0.05 37490 40357.72 2867.7 8223796.3

0.10 85817 85626.03 -191.0 36470.7

0.15 134565 130894.34 -3670.7 13473760.7

0.20 177444 176162.65 -1281.4 1641861.3

0.30 264424 266699.27 2275.3 5176854.8


28552743.8


Table 3.5.T16: Residual summary of Linearity results of impurity-E

% of

impurity

w.r.t 1000

µg/ml

Mean area

Response achieved

Response

calculated

thru Trend line

equation

Residual

(Response

practical -Response

theoretical)

Residual

square

0.05 27451 30661.76 3210.8 10308959.0

0.10 65263 65096.59 -166.4 27690.8

0.15 103809 99531.43 -4277.6 18297584.3

0.20 135244 133966.27 -1277.7 1632593.3

0.30 200325 202835.95 2510.9 6304849.5


36571676.8


3.5.7 Accuracy/Recovery

3.5.7.1 Accuracy of the assay method

Accuracy of the assay method was established by injecting three

preparations of test sample using Clopidogrel bisulfate tablets 75 mg at

50%, 100% and 150% of analyte concentration (i.e.100 µg mL-1). Each solution was injected trice (n=3) into HPLC system and the mean peak

area of Clopidogrel peak was calculated. Assay of test solution was

determined against qualified Clopidogrel bisulfate reference standard (Table 3.5.T17).

The method showed consistent and high absolute recoveries at all

three concentration (50, 100 and 150%) levels with mean absolute recovery ranging from 100.3 % to 100.8%. The obtained absolute

recoveries were normally distributed around the mean with uniform RSD

values.

3.5.7.2 Accuracy of the RS method

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

and 150% of the impurities specification limit (0.20%). Test solution prepared in triplicate (n=3) with impurities (impurity-A, impurity-B,

impurity-C, Impurity-D and impurity-E) at 0.1%, 0.20% and 0.30% level

w.r.t. analyte concentration (i.e. 1.0 mg mL-1). Mean % recovery of

impurities calculated in the test solution using the area of impurities standard at 0.20% level with respect to analyte (Table 3.5.T17).

Table 3.5.T17: Recovery results

Amount

spike

d

% Recovery Mean ± % RSD for three determinations

Clopidogrel

Impurity-A

Impurity -B

Impurity -C

Impurity –D

Impurity –E

50%

100.8±

0.35

99.1±

0.28

98.1±

0.23

98.5±

0.11

100.5±

0.15

99.9±

0.37

100

%

100.3±

0.27

97.5±

0.42

99.9±

0.33

100.1±

0.17

99.5±

0.23

98.6±

0.39

150 %

100.5± 0.33

99.1± 0.28

102.1± 0.36

98.7± 0.45

98.9± 0.29

100.5± 0.19

The related substances method showed consistent and high absolute recoveries of all five impurities at all three concentration (50,100 and

150%) levels with mean absolute recovery ranging from 97.5 % to 102.1%

in drug product.

3.5.8 Solution state stability

The solution state stability of Clopidogrel in diluent (i.e. mobile phase)

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

sample in tightly capped volumetric flasks at room temperature for two days. The same sample solution were assayed for every 12 hours interval

up to the study period, each time freshly prepared reference standard

was used to estimate the assay of test sample. The % RSD of assay of Clopidogrel during solution stability experiments was with in 1%.

The solution stability of Clopidogrel in the related substance method

was carried out by leaving sample solution in tightly capped volumetric flask at room temperature for two days. Content of Impurity-A, Impurity-

B, Impurity-C, Impurity-D and impurity-E were checked for every 12

hours interval up to the study period. No significant change was observed in the impurity content during solution stability experiments

from the initial values up to the study period. Hence Clopidogrel sample

solutions were stable for at least 48 hours in the developed method.

Table 3.5.T18: Solution stability results of the assay method

S.No Interval %Assay

Solution stability

1 0 h 100.3

2 12 h 100.4

3 24 h 99.6

4 36 h 99.8

5 48 h 100.2

% RSD 0.3

3.5.9 Robustness

To determine the robustness of the developed method experimental

conditions were purposely altered (flow rate 0.9 mL min-1 and 1.1 mL

min-1, varying ethanol content in mobile phase by +/- 3%, varying

column temperature from 25°C to 300C) and the resolution between all

pairs of compounds was greater than 2.0 and tailing factor for

Clopidogrel and its impurities was less than 1.2. The assay variability of

Clopidogrel was within ±1%. The variability in the estimation of

Clopidogrel bisulfate impurities was within ±10%.

3.5.10 Mass balance

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 samples of Clopidogrel bisulfate tablets were

assayed against the qualified reference standard and the results of mass

balance obtained were very close to 99%. The results of mass balance obtained in each condition are presented below (Table 3.5.T19).

Table 3.5.T19: Mass balance of the assay method

Stress condition Time

~% Degradation

% Assay of

active

substance

Mass balance

(%Assay +

%impurities +% Degradation

products)

Acid hydrolysis

(0.5 N HCl, 80°C ) 24 h 0.6 98.6 99.2

Base hydrolysis

(0.5 N NaOH,

80°C )

24 h 7.1 92.2 99.3

Oxidation

(3% H202) 24 h 12.8 86.1 98.9

Water hydrolysis

(80°C) 24 h 0.5 98.6 99.1

Thermal (60°C) 10 days 1.3 98.2 99.5

Photolytic

degradation 10 days 0.2 99.6 99.8

3.6 Summary and Conclusions

A new normal phase liquid chromatographic method was developed

for assay and impurities estimation of Clopidogrel bisulfate with stability-indicating power for pharmaceutical dosage forms. The proposed

developed method is capable of separating all chiral and non chiral

impurities of Clopidogrel bisulfate in presence degradation impurities formed under forced degradation studies and also proposed method can

be employed for assay determination of Clopidogrel bisulfate in

pharmaceutical dosage forms. The column used for development of this

method was Chiralcel OJ-H 250 mm x 4.6 mm, 5μm. Ethanol and n- Hexane and diethyl amine in 5:95:0.05, v/v/v; ratio was employed as a

mobile phase. The detection wavelength was to monitor eluted

components was 240 nm. Clopidogrel bisulfate tablets were exposed to

degradation conditions of acid, oxidative, base, hydrolytic, photolytic and

thermal degradation. The degradation impurities were well separated from Clopidogrel peak and its related compounds, showed the stability-

indicating power of the developed method. The developed method was

validated as per ICH guidelines for precision, accuracy, linearity, specificity, limit of detection, limit of quantification, system suitability

and robustness and found to be precise, accurate, linear rugged, robust

and specific. The developed can be employed assay and purity estimation

of Clopidogrel bisulfate drug substance (API). Thus the developed and validated normal phase liquid chromatographic method can be used for

stability analysis and production samples analysis of Clopidogrel

bisulfate in API and in pharmaceutical dosage forms.

Table 3.6.T1 Summary of analytical method validation

Test

parameter Related Substance method

Assay

method

Imprity-A

Impurity-B

Impurity-C

Impurity-D

Impurity-

E

Precision

(RSD) 1.53 2.11 1.64 1.44 1.93 0.81

LOQ

(µg mL-1) 0.410 0.320 0.310 0.360 0.360 0.320

LOD

(µg mL-1) 0.160 0.140 0.090 0.110 0.130 0.120

Precision

at LOQ(%RSD)

1.3 3.7 2.6 1.2 1.6

Linearity

(Corre

coefficient)

0.9992 0.9999 0.9991 0.9995 0.9989 0.9999

Accuracy %

at 100%

(Drug product)

97.5 99.9 100.1 99.5 98.6 100.3

Robustness

Resolution between all pairs of compounds was greater than

2.0 and tailing factor for Clopidogrel and its impurities was less than 1.2.

Solution Stability

Stadard and test solutions are stable for 48 hrs at room

temperature

Specificity

For all stress conditions, purity angle is less than the purity

threshold and there is no purity flag observed for purity

results

Reference:

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