Spectrophotometric Determination of Clopidogrel in Pharmaceutical Formulations


M. Shireesha, L. Madhavi and G. Tuljarani*

Department of Pharmaceutical Analysis, Sarojini Naidu Vanitha Pharmacy Maha Vidyalaya, Exhibition Grounds, Nampally, Hyderabad-500001 (A.P) India.

*Corresponding Author E-mail: tuljapharma@yahoo.com



Two simple extractive spectrophotometric methods are described for the determination of clopidogrel in pure and pharmaceutical formulations. The method A is based on the formation of ion association complex of the clopidogrel with orange-G in acidic buffer followed by extraction into chloroform. Method B is based on the formation of colored complex in co-ordination of secondary/ tertiary amine group of drug with cobalt thiocyanate in acidic buffer of pH 2.0 which is extractable in to nitrobenzene. The absorbance of organic layer for each method was measured at appropriate λmax against reagent blank. Both the methods obeyed Beer’s linearity in the concentration range 50-250 μg/mL for method A and 100-500 μg/mL for method B. These methods were successfully employed for the determination of clopidogrel in pharmaceutical preparations. These methods have been statistically evaluated and are found to be precise and accurate.


KEYWORDS: Clopidogrel, Orange –G, Cobalt thiocyanate, Extractive, UV-Visible Spectrophotometer.



Clopidogrel chemically is methyl 2-(2-chlorophenyl)-2-(6, 7-dihydrothieno [3, 2-c] pyridin-5(4H)-yl) acetate ( Fig.1), is a platelet aggregation inhibitor and anti thrombic, used to inhibit blood clots in coronary artery disease, peripheral vascular disease, cerebro vascular disease.


Several analytical methods like spectrophotometric1-3 methods, HPLC4-7 methods and HPTLC8 method appeared in the literature for the determination of clopidogrel in bulk and pharmaceutical formulations but there is no simple extractive spectrophotometric method. So two simple cost effective extractive spectrophotometric methods were developed for the determination of clopidogrel.



Materials and mETHOD:


An UV-Visible double beam spectrophotometer (Shimadzu 1800) with10 mm matched quartz cells was used. All weighings were done on electronic balance (Mettler balance model No.AE 200) and to adjust pH,   Systronics μ pH systems 361 were used.


Reagents and chemicals:

Pharmaceutical grade clopidogrel was provided by Aurobido Pharma Ltd, Hyderabad. Clopidogrel tablets (CLOPILET 75 mg) were procured from the local pharmacy. All the chemicals used were of analytical grade and distilled water was used for the entire work.


Preparation of reagents:

For method A:

Orange-G solution (0.2%): 200 mg of Orange-G was dissolved in 100 mL of distilled water and washed with chloroform to remove chloroform soluble impurities.

0.1M HCL solution: 0.85 mL of concentrated HCL is diluted to 100 mL with distilled water.

Chloroform: S. D fine chemicals Ltd., chloroform was used.


For method B:

Cobalt thiocyanate solution (CTC): Prepared by dissolving 7.25 gm of cobalt nitrate and 3.8 gm of ammonium thiocyanate in 100 mL of distilled water.

Buffer solution (pH 2): Prepared by mixing 25 mL of KCL (0.2 M) and 13 mL of HCL (0.2 M) and made up to 100 mL with distilled water and pH was adjusted to 2.0.

Nitrobenzene: Analytical grade nitrobenzene was used.

Preparation of standard drug solution (1mg/mL): 100 mg of clopidogrel was taken into 100 mL volumetric flask, dissolved with 10 mL of methanol and diluted to 100 mL with distilled water.


General procedure for calibration:

For method A:

Aliquots of standard clopidogrel solution (0.5 - 2.5 mL) were delivered into a series of 60 mL separating funnels. Then 1.5 mL of 0.1 M HCL and 2.5 mL of orange-G solution were added and the total volume of aqueous phase in each separating funnel was adjusted to 10 mL. To each separating funnel 10 mL of chloroform was added and the contents for shaken for 2 min. Then the two phases were allowed to separate and the absorbance of the separated chloroform layer was measured at 485 nm against the reagent blank.


For method B:

Aliquots of standard clopidogrel solution (1.0 - 5.0 mL) were delivered into a series of 60 mL separating funnels. To each separating funnel 1.0 mL of pH 2.0 buffer and 2.5 mL of CTC solution were added and the total volume of aqueous phase in each funnel was adjusted to 10 mL. Then 10 mL of nitrobenzene was added and the contents were shaken for 2 min. The two phases were allowed to separate and the absorbance of the separated nitrobenzene layer was measured at 626 nm against the reagent blank. The optimum conditions are presented in Table-1.


Table: 1 Optimum conditions and results of the proposed methods


Method A

Method B

Drug solution taken (μg/mL)

Volume of buffer (mL)

Volume of reagent (mL)

λ max(nm)











Twenty tablets were taken, weighed accurately, and powdered. Tablet powder equivalent to 100 mg of clopidogrel was weighed transferred to 100 mL volumetric flask. To dissolve the drug 10 mL of methanol was added and the volume was made up to the mark with distilled water. The solution was filtered through whatmann filter paper. After suitable dilution the solution was analysed by the above procedures.



The proposed methods for the determination of clopidogrel obeyed Beer’s law in range of 50-250 μg/mL for method A, 100-500 μg/mL for method B and molar absorptivity of 3192.461 L mol-1 cm-1 and 145.623 L mol-1 cm-1 for method A and B. Linear regression of absorbance on concentration gave the equation 0.4179x - 0.3309 with correlation coefficient (r2) 0.9936 for method A and 0.0472x - 0.0535 with correlation coefficient (r2) 0.9987 for method B. All the optical and regression characteristics are presented in Table-2.


Table: 2 Optical characteristics, precision, and accuracy of the proposed methods

Parameter                          Method A           Method B

λmax(nm)                               485                      626

Beer’s law limits (µg/mL)



Sandell’s sensitivity(mcg/cm2/0.001 A.U)



Molar absorptivity 

(L mol-1 cm-1)



Correlation coefficient(r2)



Regression equation


y = 0.4179x - 0.3309

y = 0.0472x - 0.0535







Range of errors*

Confidence limit with 0.05level

Confidence limit with 0.01level







% RSD                                               0.2775                 0.8636

(y=b+ax)*, where y is the absorbance, x is concentration in μg/ml

**Average of six determinations.



To evaluate the validity and reproducibility of the method, known amount of pure drug was added to the previously analyzed pharmaceutical preparation and the resultant mixture was analyzed. The recovery studies are summarized in Table-3. Recovery experiments indicated the absence of interferences from the commonly encountered pharmaceutical additives and excipients. The accuracy of these methods was ascertained by comparing the results of proposed methods with reference method (UV method) statistically by the T-test and F-test.  Commercial formulation of clopidogrel were successfully analysed by the proposed methods.



Table 3: Assay and recovery studies of proposed methods

Name of the dosage form

Labeled amount (mg)

Content of the drug found a(mg) ± S.D

% Recovery by the proposed method

Proposed method

Reference method

Clopidogrel Tablet (method A)










Clopidogrel Tablet (method B)







a Average ± standard deviation of eight determinations, the T and F- values referred to comparison of proposed method with reference method. Theoretical values at 95% confidence limits T = 2.365 and F=4.88

Methanol was used as solvent for reference UV method.




The developed method was found to be accurate and precise. Statistical data suggested that it could be used for the routine analysis of clopidogrel in bulk form.



The authors are thankful to Aurobindo Pharmaceuticals Ltd., Hyderabad, for providing gift samples and to the Management of Sarojini Naidu Vanita Pharmacy Maha Vidyalaya, Nampally, Hyderabad, for providing work facilities.



1.       F.A.E Yazbi, M.E.Mahrous, H.H.Hammud, G.M.Sonji, N.M.Sonji. Kinetic spectrophotometric determination of betoxolol, clopidogrel and imidapril in pharmaceutical preparations. Current. Analytical Chemistry. 2010; 6: 228-236.

2.       Chaudhari Pritam B, Pawar Pravin D, Narkhede Kiran P. Stability indicating spectrophotometric method for determination and validation of clopidogrel bisulphate in tablet dosage form. International Journal of Research in Ayurveda and Pharmacy. 2010; 1: 418-423.

3.       B. Anupama, V. Jagathi, A.Aparna, M.Madhu babu, V.Lakshmi Annapurna. Assay of clopidogrel by visible spectrophotometry. International Journal of Pharma and Biosciences. 2011; 2: 105-108.

4.       Sultana N, Arayne MS, Ali KA, Nawaz M. Simultaneous determination of clopidogrel and aspirin by RP-HPLC from bulk material and dosage formulations using multivariate calibration technique. Journal of Chromatographic Sciences. 2011; 49: 165-169.

5.       Effat Souri, Hassan Jaalizadeh, Abbas Kebriaee-Zadeh, Maral Shekarchi, Afshin Dalvandi. Validated HPLC method for determination of carboxylic acid metabolite of clopidogrel in human plasma and its application to a pharmacokinetic study. Biomedical Chromatography. 2006; 20: 1309-1314.

6.       Golbarg Ghiasi, Amir Farshchi, Gholamreza Bachrami. High performance liquid chromatographic determination of inactive carboxylic acid metabolite of clopidogrel in human serum. Iranian Journal of Pharmaceutical Sciences. 2009; 5: 231-238.

7.       Sagar S. Panda. Ion pairing RP-HPLC method for simultaneous determination of aspirin and clopidogrel bisulphate in tablet and capsule dosage form. International Journal of PharmaTech Research. 2010; 2: 269-273.

8.       Purushotham K. Sinha, Mrinalini C. Danule and K.G.Bothara. A validated stability indicating HPTLC method for determination of aspirin and clopidogrel bisulphate in combined dosage form. Eurasian Journal of Analytical Chemistry. 2009; 4: 152-160.




Received on 20.07.2011        Modified on 05.08.2011

Accepted on 14.08.2011        © AJRC All right reserved

Asian J. Research Chem. 4(10): Oct., 2011; Page 1566-1568