Experimental design approach for the development and validation of an enantiospecific RP-HPLC method for simultaneous determination of clopidogrel and related compounds

Rumenka Petkovska, Claus Cornett, Aneta Dimitrovska


An enantiospecific RP-HPLC method was developed and validated for the simultaneous determination of clopidogrel and four related compounds specified as impurities. Experimental design was applied during the method optimization (Full factorial 23 design) and robustness testing (Central Composite Face Centered design). Laboratory mixtures of clopidogrel and its impurities in a concentration ratio of 1: 5.0×10–4 were used as an investigation matrix. The three independent variables were the acetonitrile content in the mobile phase, pH of the mobile phase, and the column temperature. A Chromatographic Response Function (CRF) was used for estimation of the system response resolution (Rs). Separation was achieved using mobile phase composition of ACN: Buffer solution pH 6.5 (40:60 v/v) at 30 ºC. A CHIRAL-AGP 4.0 mm × 100 mm, 5.0 μm particle size column was used. The total time for chromatographic separation was approximately 10.0 min. The method was validated for its selectivity, linearity, precision, accuracy and robustness.


experimental design; enantiomer separation; clopidogrel; impurities; method development; validation

Full Text:



B. Jarvis, K. Simpson, Clopidogrel: A Review of its Use in the Prevention of Atherothrombosis, Drugs, 60 (2), 347–377 (2000).

K. Moshfegh, M. Redondo, F. Julmy, W. A. Wuillemin, M. U. Gebauer, A. Haeberli, and B. J. Meyer, Antiplatelet effects of clopidogrel compared with aspirin after myocardial infarction: enhanced inhibitory effects of combination therapy, J. Am. Coll. Cardiol., 36 (3), 699–705 (2000).

T. L. Lenz, A. F. Wilson, Clinical Pharmacokinetics of Antiplatelet Agents Used in the Secondary Prevention of Stroke, Clin. Pharmacokinet., 42, 909–920, (2003).

A. Mitakos, I. Panderi, A validated LC method for the determination of clopidogrel in pharmaceutical preparations, J. Pharm. Biomed. Anal., 28, 431–438 (2002).

Y. Gomez, E. Adams, J. Hoogmartens, Analysis of purity in 19 drug products tablets containing clopidogrel: 18 copies versus the original brand, J. Pharm. Biomed. Anal., 34, 341–348 (2004).

H. Ksycinska, P. Rudzki, M. Bukowska-Kiliszek, Determination of clopidogrel metabolite (SR26334) in human plasma by LC-MS, J. Pharm. Biomed. Anal., 41, 533– 539 (2006).

A. Mitakos, I. Panderi, Determination of the carboxylic acid metabolite of clopidogrel in human plasma by liquid chromatography-electrospray ionization mass spectrometry, Anal. Chim. Acta, 505, 107–114, (2004).

S. S. Singh, K. Shama, D. Barot, P. R. Mohan, V. B. Lohray, Estimation of carboxylic acid metabolite of clopidogrel in Wistar rat plasma by HPLC and its application to a pharmacokinetic study, J. Chromatogr. B, 821, 173– 180 (2005).

International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Topic Q3A (R2), Impurities in New drug substances (2006)

N. Matthijs, C. Perrin, M. Maftouh, D. L. Massart, Y. Vander Heyden, Definition and system implementation of strategies for method development of chiral separation in normal-or reversed-phase liquid chromatography using polysaccharide-based stationary phases, J. Chromatogr. A, 1041, 119–133, (2004).

T. Lundsted, E. Seifert, L. Abramo, B. Thelin, A. Nystorm, J. Pettersen and R. Bergman, Experimental design and optimization. Chemom. Intell. Lab. Syst., 42, 3–40, (1998).

European Pharmacopeia, 6th Edition, Concil of Europe, Strasbourg, 2007, pp.74.

P. W. Araujo, R. G. Brereton, Experimental design, II. Optimization. Trends Anal Chem., 15, 63–68 (1996).

S. N. Deming and S. L. Morgan, Experimental design: A chemometric approach, Elsevier, Amsterdam, Netherlands. 1993.

D. Bylund, A. Bergens and S. P. Jacobsson, Optimization of Chromatographic Separations by Use of a Chromatographic Response Function, Empirical Modeling and Multivariate Analysis. Chromatographia, 44, 74–80 (1997).

International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use. Topic Q2 (R1), Validation of Analytical Procedures: Text and Methodology, 2006.

H. Fabre, Robustness testing in liquid chromatography and capillary electrophoresis. J. Pharm. Biomed. Anal., 14, 1125–1132 (1996).

G. A. Lewis, D. Mathieu and R. Phan-Tan-Luu, Pharmaceutical Experimental Design, Marcel Decker, New York USA 1999.

DOI: http://dx.doi.org/10.20450/mjcce.2008.247


  • There are currently no refbacks.

Copyright (c) 2016 Rumenka Petkovska, Claus Cornett, Aneta Dimitrovska

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.