Development and Validation of Analytical Method for Clopidogrel Bisulphate and Irbesartan by Simultaneous Equation Spectroscopic Method

 

Pankaj Savani1*, Sudhanshu Chauhan1, Vineet Jain2, Hasumati Raj1, Sagar Patel1

Department of Quality Assurance, Shree Dhanvantary Pharmacy College, Kim, Dist: Surat, Gujarat, India. 394110

2Department of Pharmacognosy, Shree Dhanvantary Pharmacy College,  Kim, Dist: Surat, Gujarat, India. 394110

*Corresponding Author E-mail: pankajsavani75@gmail.com

 

ABSTRACT:

A simple, accurate and precise spectroscopic method was developed for simultaneous estimation of Clopidogrel Bisulphate and Irbesartan in synthetic mixture using simultaneous equation Method. In this spectroscopic method, for Clopidogrel Bisulphate 220.00 nm and 250.00 nm wavelengths were selected for measurement of absorptivity. Both the drugs show linearity in a concentration range of 10-50 μg/ml at their respective λmax with correlation coefficient (r2) of 0.9996 and 0.9998 for Clopidogrel Bisulphate and Irbesartan, respectively. Accuracy, precision and recovery studies were done by QC samples covering lower, medium and high concentrations of the linearity range. The relative standard deviation for accuracy, precision studies were found to be within the acceptance range (< 2%). The limit of determination was 0.243 μg/ml and 0.323 μg/ml for Clopidogrel Bisulphate and Irbesartan, respectively. The limit of quantification was 0.737 μg/ml and 0.980 μg/ml for Clopidogrel Bisulphate and Irbesartan, respectively. Recovery of Clopidogrel Bisulphate and Irbesartan were found to be 99.57 % and 99.68 % respectively confirming the accuracy of the proposed method.  % Assay was found to be 99.41 % and 99.22 % for Clopidogrel Bisulphate and Irbesartan, respectively. The proposed method is recommended for routine analysis since they are rapid, simple, accurate and also sensitive and specific by no heating and no organic solvent extraction.

 

KEYWORDS: Clopidogrel Bisulphate, Irbesartan, simultaneous estimation, Validation method.

 

 

INTRODUCTION:

The present study was aimed to develop simple, rapid, accurate and precise analytical method for simultaneous estimation of Clopidogrel Bisulphate (CLO) and Irbesartan (IRB). Clopidogrel Bisulphate and Irbesartan are two widely used antithrombogenic agents and co-administration produces an enhanced therapeutic effect in many clinical conditions, particularly in high-risk patients with acute coronary syndromes and renal injury.(1)

 

Antiplatelet drugs are used in platelet function of prophylaxis and thromboembolic disorders.(2) They are therapeutic rather than prophylactic and work by activating the natural fibrinolytic system. Clopidogrel Bisulphate inhibits the ADP receptor blockers. As Fibrinogen Thienopyridine surface receptor on platelets is Inhibits selective irreversible P2Y12 Purinergic Receptor So Inhibits ADP, fibrinogen induced platelet Aggregation and Adenylyl cyclase then result to reduce inflammation. IUPAN name of Clopidogrel Bisulphate Methyl 2- (2- Chlorophenyl) -2- (6,7- dihydro thieno [3.2-C] Pyridine- 5 (4H)-yl) Acetate sulphate.(3) Clopidogrel Bisulphate is white crystalline powder. Solubility is given in practically insoluble in water, soluble in methanol, slightly soluble in 0.1 N HCl.(4)

 

 

Figure 1: Structure of Clopidogrel Bisulphate

 

Irbesartan is an Angiotensin II receptor Antagonist. It used mainly for the treatment of hypertension. It is an orally active nonpeptide tetrazole derivative and selectively inhibits Angiotensin II receptor type 2. Hypertension is the most common cardiovascular condition and its result to increased peripheral vascular smooth muscle tone, which leads to increased arteriolar resistance and reduced capacitance of the venous system. Angiotensin II receptor type1 antagonists have been widely used in treatment of diseases like hypertension, heart failure, cerebrovascular accidents, myocardial infarction and diabetic nephropathy. IUPAN name of Irbesartan is 3-[(2’- (2H-tetrazol-5-yl) - (1,1’-biphenyl) -4-yl) methyl] -2- butyl-1,3-diazolspiro (4,4) non-1-en-4-one.(5) Irbesartan is white or almost white, crystalline powder. Solubility is given in practically insoluble in water, freely soluble in methanol, slightly soluble in 0.1 N NaOH.(6)

 

 

Figure 2: Structure of Irbesartan

 

Combination effect of Genetic Polymorphism on Clopidogrel Efficacy and Cardiovascular Events in the Clopidogrel in the Unstable Angina to Prevent Recurrent Events Trial and the A trial Fibrillation Clopidogrel Trial with Irbesartan for Prevention of Vascular Events.(7) Irbesartan in used in lower blood pressure in hypertension but its combination of Antiplatelet drug such as a Clopidogrel Bisulphate to used in Anti-inflammatory Renoprotective effect of Chronic renal injury. Platelet activation and Angiotensin-II may each contribute to glomerular inflammation and fibrosis.(8)

The review of literature regarding quantitative analysis of Clopidogrel Bisulphate and Irbesartan revealed that no attempt was made to develop analytical methods for Clopidogrel Bisulphate and Irbesartan. Some spectrometric methods and chromatographic methods have been reported for the estimation of the individual drugs. The focus of the present study was to develop and validate a rapid, stable, specific, and economic high performance liquid chromatographic method for the estimation of Clopidogrel Bisulphate and Irbesartan in Synthetic mixture.(9)

 

MATERIALS AND METHODOLOGY:

Apparatus and Instrument:

A double beam UV-Visible spectrophotometer (Shimadzu model 2450, Japan) with spectral width of 2.0 nm, 1.0 cm quartz cells was used to measure absorbance of all the solutions.

 

·      Spectra were automatically obtained by UV-Probe system software.

·      An analytical balance (Sartorius CD 2250, Gottingen, Germany) was used for weighing purpose.

·      Sonicator (D120/2H, Trans-O-Sonic) was used Sonication of solution.

·      All instruments and glass wares were calibrated.

 

REAGENTS AND MATERIAL:

Clopidogrel Bisulphate raw material was received as gift sample from Cadila Healthcare LTD, Ankleshwar.  Irbesartan raw material was received as gift sample from CTX Life Science, Surat. Methanol AR Grade (FINAR), Distilled water, HCl AR Grade, NaOH AR Grade (Ranchem) was used for development purpose.

 

Standard solution of Clopidogrel Bisulphate (CLO):

Preparation of stock solution of CLO:

An accurately weighed quantity of Clopidogrel Bisulphate (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved, diluted up to mark with methanol to obtain standard solution having concentration of Clopidogrel Bisulphate (100 μg/ml).

 

Standard solution of Irbesartan (IRB):

Preparation of standard stock solution of IRB:

An accurately weighed quantity of  Irbesartan (10 mg) was transferred to a separate 100 ml volumetric flask and dissolved, diluted up to mark with methanol to obtain standard solution having concentration of Irbesartan (100 μg/ml).

 

Preparation of Standard Mixture Solution (CLO + IRB):

1.0 ml of standard stock solution of CLO (100 μg/ml) and 1.0 ml of standard stock solution of IRB (100 μg/ml) were pipetted out into 10 ml volumetric flasks and volume was adjusted to the mark with methanol to get 10 μg/ml of CLO and 10 μg/ml of IRB.

 

Preparation of Test Solution:

The preparation of synthetic mixture was as per patent(10)

 

Table 1: Composition of formulation (Synthetic Mixture)

Sr. No.

Drug

Quantity (mg)

Quantity

(mg)

1

Clopidogrel Bisulphate

150

1500

2

Irbesartan

150

1500

3

Lactose

72.5

725

4

Starch

48

480

5

Anhydrous Silica

0.5

5.0

6

Magnesium Stearate

4.0

40

7

Talc

q.s.

q.s.

 

Total

450

4500

 

Above all ingredients were shift and blend to make uniformity of mixing. Take synthetic powder equivalent to 10 mg of Clopidogrel Bisulphate in 100 ml volumetric flask. Dissolve in 25 ml of Methanol and Sonicated for 15 min. Dilute up to 100 ml with solvent shake vigorously. Filtered through Whatman filter paper No. 42 and further diluted. Finally the solution had concentration of 100 μg/ml for CLO and IRB, respectively. From that pipette out 1.0 ml in 10 ml volumetric flask and volume was made up to mark with Methanol to make final concentration of mixture 10 µg/ml for CLO and IRB, respectively.

 

Calibration curves for Clopidogrel Bisulphate:

This series consisted of five concentrations of standard CLO solution ranging from 10 to 50 μg/ml. The solutions were prepared by pipetting out Standard CLO stock solution (100 μg/ml). Then pipetting out (1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml and 5.0 ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol. A zero order spectrum of the resulting solution was recorded, measured the absorbance at 220.00 nm against a reagent blank solution (methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of CLO (Figure 3).

 

Calibration curve for Irbesartan:

This series consisted of five concentrations of standard IRB solution ranging from 10 to 50 μg/ml. The solutions were prepared by pipetting out Standard IRB stock solution (1.0 ml, 2.0 ml, 3.0 ml, 4.0 ml and 5.0 ml) was transferred into a series of 10 ml volumetric flask and volume was adjusted up to mark with methanol. A zero order spectrum of the resulting solution was recorded, measured the absorbance at 250.00 nm against a reagent blank solution (methanol). Calibration curve was prepared by plotting absorbance versus respective concentration of IRB (figure 3).

 

Development and Validation of Spectroscopic Simultaneous Equation Method(11)

Selection of wavelength and method development for determination of Clopidogrel Bisulphate and Irbesartan

The standard solution of CLO and IRB were scanned separately between 200-400 nm, and CLO showed absorbance maxima at 220.00 nm and IRB at 250.00 nm. (Figure 3)

 

 

 

Figure 3: overlain zero order spectra of CLO and IRB (Ratios: 1:1)

 

VALIDATION PARAMETERS:(12)

1.    Linearity and Range

The Zero order (figure 3) showed linear absorbance at 220.00 nm for CLO (10-50 µg/ml) and 250.00 nm for IRB (10-50 µg/ml) with correlation coefficient (r2) of 0.9996 and 0.9998 for CLO and IRB, respectively.

 

 

This method obeyed beer’s law in the concentration range 10-50 µg/ml CLO and IRB, respectively. (Table 2) Correlation coefficient (r2) for calibration curve of CLO and IRB was found to be 0.9996 and 0.9998, respectively (figure 3 and 7) The regression line equation for CLO and IRB are as following,

y = 0.028x + 0.048 for CLO _____________ (1)

y = 0.037x - 0.030 for IRB ______________ (2)

Table 2: Calibration data for CLO at 220.00 nm and 250.00 nm *(n=6)

Sr. No.

Concentration (μg/ml)

Abs.* + SD CLO (220.00 nm)

% RSD

Abs.* + SD CLO (250.00 nm)

% RSD

1

10

0.3140 + 0.0028

0.918

0.0918 + 0.0007

0.819

2

20

0.6221 + 0.0038

0.623

0.1450 + 0.0010

0.713

3

30

0.8990 + 0.0032

0.363

0.2008 + 0.0019

0.966

4

40

1.1681 + 0.0023

0.198

0.2435 + 0.0018

0.771

5

50

1.4423 + 0.0033

0.234

0.2836 + 0.0028

0.988

 

Table 3: Calibration data for IRB at 220.00 nm and 250.00 nm *(n=6)

Sr. No.

Concentration (μg/ ml)

Abs.* + SD IRB (220.00 nm)

% RSD

Abs.* + SD IRB (250.00 nm)

% RSD

1

10

0.5811 + 0.0033

0.582

0.3542 + 0.0023

0.654

2

20

1.1970 + 0.0021

0.178

0.7110 + 0.0035

0.492

3

30

1.8723 + 0.0058

0.310

1.0991 + 0.0041

0.375

4

40

2.4338 + 0.0024

0.102

1.4928 + 0.0038

0.259

5

50

2.8692 + 0.0046

0.161

1.8508 + 0.0033

0.178

 

Table 4: Average of absorptivity at 220.00 nm and 250.00 nm

at 220 nm

at 250 nm

ax1

0.03010

ax2

0.00695

ay1

0.06005

ay2

0.03638

 

 

Figure 4: Calibration curve for CLO at 220.00 nm

 

 

Figure 5: Calibration curve for CLO at 250.00 nm

 

 

Figure 6: Calibration curve for IRB at 220.00 nm

 

 

Figure 7: Calibration curve for IRB at 250.00 nm

 

 

 

 

Figure 8: Zero order spectra of CLO and IRB in combination (1:1 ratio)

Table 5: Mixture linearity for CLO and IRB (1:1 ratio) 

Sr. No.

Concentration in mixture (μg/ml)

Abs. + SD  CLO (220.00 nm)

% RSD

Abs. + SD  IRB  (250.00 nm)

% RSD

CLO

IRB

1

10

10

0.9925 + 0.0093

0.946

0.4695 + 0.0095

0.900

2

20

20

1.6095 + 0.0077

0.479

0.8791 + 0.0085

0.974

3

30

30

2.1716 + 0.0087

0.402

1.3681 + 0.0093

0.683

4

40

40

2.6813 + 0.0079

0.296

1.8293 + 0.0080

0.438

5

50

50

3.1891+ 0.0091

0.285

2.2851 + 0.0089

0.389

 

 

 

 

Figure 9: Calibration curve for CLO and IRB in combination

 

Precision:

Intraday precision:

The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations 10, 30, 50 μg/ml for CLO and IRB, respectively. Three replicate (n=3) each on same day. Intraday precision data presented in Table 6. These % RSD value was found to be less than 1.0 indicated that the method is precise.

 

 

Table 6: Intraday precision data for estimation of CLO and IRB *(n=3)

Conc. (μg/ml)

CLO

Abs.* + SD

% RSD

IRB

Abs.* + SD

% RSD

CLO

IRB

10

10

0.987 + 0.0030

0.309

0.466 + 0.0037

0.811

30

30

2.169 + 0.0060

0.277

1.367 + 0.0045

0.335

50

50

3.187 + 0.0055

0.174

2.283 + 0.0055

0.243

 

Interday Precision:

The precision of the developed method was assessed by analyzing combined standard solution containing three different concentrations 10, 30, 50 μg/ml for CLO and IRB, respectively triplicate (n=3) per day for consecutive 3 days for inter-day precision. Interday precision data presented in Table 7. These % RSD value was found to be less than 1.0 indicated that the method is precise.

 

Table 7: Interday precision data for estimation of CLO and IRB *(n=3)

Conc. (μg/ml)

CLO

Abs.* + SD

% RSD

IRB

Abs.* + SD

% RSD

CLO

IRB

10

10

0.994 + 0.0045

0.461

0.468 + 0.0043

0.931

30

30

2.171 + 0.0060

0.276

1.368 + 0.0055

0.402

50

50

3.189 + 0.0062

0.195

2.285 + 0.0065

0.286

 

Accuracy:

Accuracy of the method was determined by recovery study from synthetic mixture at three levels (80%, 100%, and 120%) of standard addition. The % recovery values are tabulated in Table 8 and 9. Percentage recovery for CLO and IRB by this method was found in the range of 99.23 to 100.16 % and 99.34 to 100.25 %, respectively, The value of % RSD within the limit indicated that the method is accurate and percentage recovery shows that there is no interference from the excipients.

 

 

Table 8: Recovery data of CLO *(n=3)

Concentration of CLO from formulation (mg)

Concentration of Std. CLO added (mg)

Total Concentration of CLO (μg/ml)

Total amount of CLO found (μg/ml) Mean* + SD

% Recovery (n=3)

% RSD CLO

20

-

20

19.84 + 0.080

99.23

0.407

20

16

36

35.90 + 0.160

99.37

0.996

20

20

40

39.91 + 0.081

99.55

0.411

20

24

44

44.04 + 0.050

100.16

0.209

 

Table 9: Recovery data of IRB *(n=3)

Concentration of IRB from formulation (mg)

Concentration of Std. IRB added (mg)

Total Concentration of IRB (μg/ml)

Total amount of IRB found (μg/ml) Mean* + SD

% Recovery (n=3)

% RSD IRB

20

-

20

19.92 + 0.045

99.63

0.226

20

16

36

35.92 + 0.045

99.52

0.281

20

20

40

40.05 + 0.065

100.25

0.327

20

24

44

43.84 + 0.066

99.34

0.277

 

Limit of Detection and Quantization:

The LOD for CLO and IRB was conformed to be 0.243 µg/ml and 0.323 µg/ml, respectively. The LOQ for CLO and IRB was conformed to be 0.737 µg/ml and 0.980 µg/ml, respectively. The obtained LOD and LOQ results are presented in Table 10.

 

Table 10: LOD and LOQ data of CLO and IRB *(n=10)

Conc. (μg/ml)

Abs.* + SD CLO

% RSD

Abs.* + SD IRB

% RSD

CLO

IRB

10

10

0.9854 + 0.0020

0.209

0.4645 + 0.0036

0.781

LOD (μg/ml)

0.243

0.323

LOQ (μg/ml)

0.737

0.980

ROBUSTNESS AND RUGGEDNESS:

The obtained Ruggedness and Robustness results are presented in table 11. The % RSD was found to be 0.243 - 0.979 % for CLO and 0.495 - 0.984 % for IRB. These % RSD value was found to be less than 1.0 indicated that the method is precise. No significant changes in the spectrums  were  observed,  proving  that the developed method is rugged and robust.

 

 

 

 

Table 11: Robustness and Ruggedness data of CLO and IRB *(n=3)

Robustness data of Clopidogrel Bisulphate and Irbesartan (10 μg/ml) (n=3)*

No

Factor

Level

CLO

Abs.* + SD

%RSD

IRB Abs.* + SD

%RSD

1.

Change in Instrument

UV-2450

0.992 + 0.0025

0.253

0.474 + 0.0045

0.966

UV-1800

0.994 + 0.0035

0.353

0.471 + 0.0043

0.925

2.

Change in Analyst

Analyst-1

0.991 + 0.0040

0.407

0.467 + 0.0030

0.653

Analyst-2

0.994 + 0.0030

0.301

0.470 + 0.0041

0.885

Ruggedness data of Clopidogrel Bisulphate and Irbesartan (n=3)*

3.

Change in solvent

90 % Methanol

1.604 + 0.0157

0.979

0.886 + 0.0086

0.972

10 % Methanol

1.554 + 0.0145

0.934

0.806 + 0.0079

0.984

 

 

CLO-218 nm

CLO-222 nm

IRB-248 nm

IRB-252 nm

4.

Change in Wavelength

1.034 + 0.0025

0.243

0.967 + 0.0035

0.363

0.508 + 0.0025

0.495

0.437 + 0.0036

0.825

 

Application of the proposed method for Analysis of CLO and IRB in synthetic mixture:

A zero order spectrum of the sample solution containing 20 µg/ml of CLO and 20 µg/ml of IRB was recorded and the absorbance at 220.00 nm and 250.00 nm were noted for estimation of CLO and IRB, respectively.  The concentration of CLO and IRB in mixture was determined using the corresponding calibration graph. The results from the analysis of synthetic mixture containing Clopidogrel Bisulphate (20 μg/ml) and Irbesartan (20 μg/ml) in combination are presented in Table in 12. The percentage assay shows that there is no interference from excipients and the proposed method can successfully applied to analysis of commercial formulation containing CLO and IRB. The % assay values are tabulated in Table 12.

 

Table 12: Analysis data of synthetic mixture *(n=3)

Sr. No.

Drug

Formulation (synthetic mixture) (μg /ml)

% Assay* ± SD

USP limit (%)

1

CLO

20

99.41 + 0.401

97-101.5 %

2

IRB

20

99.22 + 0.152

98-102 %

SUMMARY OF VALIDATION PARAMETER:

Table13: Summary of validation parameters

Sr. No.

PARAMETER

Clopidogrel Bisulphate

Irbesartan

1

Wavelength Max.

220.00 nm

250.00 nm

2

Linearity (µg/ml) (n=6)

10 to 50 µg/ml

10 to 50 µg/ml

3

Regression equation

y = 0.028x + 0.048

y = 0.037x - 0.030

4

Correlation coefficient (r2)

0.9996

0.9998

5

Intraday Precision (% RSD) (n=3)

0.174-0.309

0.243-0.811

6

Interday Precision (% RSD) (n=3)

0.195-0.461

0.286-0.931

7

Accuracy (% Recovery) (n=3)

99.23-100.16

99.34-100.25

8

LOD (µg/ml) (n=10)

0.243

0.323

9

LOQ (µg/ml) (n=10)

0.737

0.980

10

Robustness and Ruggedness

(% RSD) (n=3)

0.243-0.979

0.495-0.984

11

Assay

99.41

99.22

 

 

CONCLUSION:

A new, Simultaneous Equation method has been developed for estimation of Clopidogrel Bisulphate and Irbesartan in synthetic mixture. The method was validated by employment of ICH guidelines. The result of linearity, accuracy, precision proved to be within limits with lower limits of detection and quantification. Ruggedness and Robustness of method was confirmed as no significant were observed on analysis by subjecting the method to slight change in the method condition.  Assay results obtained by proposed method are in fair agreement.

 

REFERANCE:

1.     Tu X et al. Anti-inflammatory Renoprotective effect of Clopidogrel and Irbesartan in Chronic renal injury. Journal of American Society Nephrol. 19;2008:77-83.

2.     Tripathi KD. Essentials of Medical Pharmacology. Jaypee Brothers Medical Publishers (P) Limited, New Delhi. 2013.

3.     Clopidogrel Bisulphate Drug Info in drug bank (Database Available on Internet) Available from: http://www. Drugbank.ca/drugs/DB00758.

4.     Clopidogrel Bisulphate Drug Info (Database Available on Internet) Available from:  http://www.wikipedia.org/wiki/Clopidogrel.

5.     Goodman and Gilman’s. The pharmacological Basis of Therapeutics. Medical Publishing Division, New Delhi. 2001.

6.     Irbesartan Drug Info in drug bank (Database Available on Internet) Available from: http://www.Drugbank.ca/drugs/DB01029.

7.     Irbesartan Drug Info (Database Available on Internet) Available from: http://www.wikipedia.org/wiki/Irbesartan.

8.     Pare G, Ross S, Mehta SR and Salim Y. Effect of Genetic Polymorphism on Clopidogrel Efficacy and Cardiovascular Events in the Clopidogrel in the Unstable Angina to Prevent Recurrent Events Trial and the Atrial Fibrillation Clopidogrel Trial With Irbesartan for Prevention of Vascular Events. Circular Cardiovascular Genetics. 5;2012:250-256.

9.     Virani P, Sojitra R, Raj H, Jain V. Irbesartan: A review on analytical method and its determination in pharmaceuticals and biological matrix. Inventi Rapid: Pharm Analysis and Quality Assurance. 4;2014:1-6.

10.   Catherine C, Herbert, Marc J, Nisato and Dino. Pharmaceutical composition containing an Angiotensin II AT1 receptor antagonist and an Antiplatelet agent. Available from: URL: https://patents.google.com/patent/WO2000016773A1/en.

11.   Davidson AG, Beckett AH and Stenlake JB. Practical Pharmaceutical Chemistry, Published by CBS, New Delhi. 2002, pp 275.

12.   International Conference on Harmonization, Harmonized Tripartite Guideline, Validation of Analytical Procedures Text and Methodology, ICH Q2(R1), 2005.

 

 

 

Received on 16.03.2016       Accepted on 25.04.2016     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2016; 6(2): 102-108.

DOI: 10.5958/2231-5675.2016.00015.6