Determination of Clopidogrel Carboxylic Acid in Human Plasma by LC-MS/MS

Abstract

Background: Clopidogrel, is a thienopyridine derivative labeled for use to prevent thrombosis after coronary artery stenting. Pharmacokinetics of clopidogrel is studied indirectly by quantification of carboxylic acid which is a major metabolite of Clopidogrel. Objective: The aim of this work is to develop and validate a rapid, simple and sensitive LC/MS/MS assay method for the determination of Clopidogrel carboxylic acid in human plasma using Clopidogrel-D4-carboxylic acid as internal standard. Methods: Analytes was extracted from 200 μl of plasma by a simple liquid-liquid extraction using diethyl ether – n-hexane (80:20, v/v). The chromatographic separations were achieved on a C18 column using Methanol, de-ionized water and formic acid as a mobile phase at flow rate of 0.5 ml/minute. Analysis was monitored by multiple reactions monitoring mode based on m/z transition of 308.10→113 for Clopidogrel carboxylic acid and 312.10→129 for internal standard. Result: The method had a total run time of about 4 minutes. The lower limit of quantification (LLOQ) was 25 ng/ml showing good linearity over the working range of 25 – 3000 ng/ml (r ≥ 0.999). The intra- and inter day accuracies were 90% - 98% and 92.138% - 96.889% respectively (deviation within acceptable range ≤ 10%).Conclusion: It was shown that this method is suitable for pharmacokinetic study following oral administration of Clopidogrel and can be successfully applied to the therapeutic drug monitoring of Clopidogrel in Clopidogrel-treated patients.

Share and Cite:

M. El-Sadek, S. Moustafa, H. Kadi and A. Al-Hakami, "Determination of Clopidogrel Carboxylic Acid in Human Plasma by LC-MS/MS," American Journal of Analytical Chemistry, Vol. 2 No. 4, 2011, pp. 447-455. doi: 10.4236/ajac.2011.24054.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] D. C. Mills, R. Puri, C. J. Hu, C. Minniti, G. Grana, M. D. Freedman, R. F. Colman and R. W. Colman, “Clopidogrel Inhibits the Binding of ADP Analogues to the Receptor Mediating Inhibition of Platelet Adenylate Cyclase,” Arteriosclerosis Thrombosis, Vol. 12, No. 4, 1992, pp. 430-436. doi:10.1161/01.ATV.12.4.430
[2] P. W. Majerus and D. M. Tollefsen, “Blood Coagulation and Anticoagulant, Thrombolytic, and Antiplatelet Drugs,” In: L. L. Brunton, Ed., The Pharmacological Basis of Therapeutics, 11th Edition, The McGraw-Hill Companies, New York, 2006, p. 1483.
[3] PLAVIXTM, 2011. www.RXlist.com
[4] Safety, 2011.http://www.fda.gov/medwatch/safety/2007/May_PI/Plavix_PI.pdf
[5] P. Savi, J. Combalbert, C. Gaich, M. C. Rouchon, J. P. Maffrand, Y. Berger and J. M. Herbert, “The Antiaggre-gating Activity of Clopidogrel Is Due to a Metabolic Ac-tivation by the Hepatic Cytochrome P450-1A,” Thromb Haemostasis, Vol. 72, 1994, pp. 313-317.
[6] J. M. Pereillo, M. Maftouh, A. Andrieu, M. F. Uzabiaga, O. Fedeli, P. Savi, M. Pascal, J. M. Herbert, J. P. Maf-frand and C. Picard, “Structure and Stereochemistry of the Active Metabolite of Clopidogrel,” Drug Metabolism and Disposition, Vol. 30, No. 11, 2002, pp. 1288-1295. doi:10.1124/dmd.30.11.1288
[7] Y. Gomez, E. Adams and J. Hoogmartens, “Analysis of Purity in 19 Drug Product Tablets Containing Clopidogrel: 18 Copies Versus Theoriginal Brand,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 34, No. 2, 2004, pp. 341-348.
[8] J. M. Herbert, D. Frehel, E. Vallee, G. Kieffer, D. Gouy, Y. Berger, G. Defreyn and J. P. Maffrand, “Clopidogrel, a Novel Antiplateletand Antithrombotic Agent,” Cardi-ovascular Drugs, Vol. 11, No. 2, 1993, pp. 180-198.
[9] T. R. Rao, P. R. Usha, M. U. Naidu, J. A. Gogtay and M. Meena, “Bioequivalence and Tolerability Study of Two Brands of Clopidogrel Tablets, Using Inhibition of Platelet Aggregation and Pharmacodynamic Measures,” Current Therapeutic Research, Vol. 64, No. 9, 2003, pp. 685- 696. doi:10.1016/j.curtheres.2003.09.014
[10] H. Ksycinska, P. Rudzki and M. Bukowska-Kiliszek, “Determination of Clopidogrel Metabolite (SR26334) in Human Plasma by LC–MS,” Journal of Pharmaceutical and Biomedical Analysis, Vol. 41, No. 2, 2006, pp. 533- 539. doi:10.1016/j.jpba.2005.11.035
[11] P. C. A. Kam and C. M. Nethery, “The Thienopyridine Derivatives (Platelet Adenosine Diphosphate Receptor Antagonists), Pharmacology and Clinical Developments,” Anaesthesia, Vol. 58, No. 1, 2008, pp. 28-35. doi:10.1046/j.1365-2044.2003.02960.x
[12] J. McEwen, G. Strauch, P. Perles, G. Fowter, T. Moreland, J. P. Dickinson, R. Meontels, J. Mosser and J. Neccian, “Clopidogrel Bioavailability Is Unaffected by Food or Antacids,” The Journal of Clinical Pharmacology, Vol. 36, No. 9, 1996, p. 856.
[13] G. Vilahur, B. G. Choi, M. U. Zafar, J. F. Viles-Gonzalez, D. A. Vorchheimer, V. Fuster and J. J. Badimon, “Nor-malization of Platelet Reactivity in Clopidogrel-Treated Subjects,” Journal of Thrombosis and Haemostasis, Vol. 5, No. 1, 2007, pp. 82-90. doi:10.1111/j.1538-7836.2006.02245.x
[14] R. H. Hongo, J. Ley, S. E. Dick and R. R. Yee, “The Effect of Clopidogrel in Combination with Aspirin When Given before Coronary Artery Bypass Grafting,” Journal of the American College of Cardiology, Vol. 40, No. 2, 2002, pp. 231-237. doi:10.1016/S0735-1097(02)01954-X
[15] K. A. Kim, P. W. Park and J. Y. Park, “Effect of CYP- 3A5*3 Genotype on the Pharmacokineticsand Antiplatelet Effect of Clopidogrel in Healthy Subjects,” European J ournal of Clinical Pharmacology, Vol. 64, No. 6, 2008, pp. 589-597. doi:10.1007/s00228-008-0471-0
[16] R. V. S. Nirogi, V. N. Kandikere, M. Shukla, K. Mudi-gonda, S. Maurya and R. Boosi, “Quantification of Clo-pidogrel in Human Plasma by Sensitive Liquid Chroma-tography/Tandem Mass Spectrometry,” Mass Spectrome-try, Vol. 20, No. 11, 2006, pp. 1695-1700.
[17] R. A. Kulkarni, “Clopidogrel in Cardiovascular Disord-ers,” Journal of Postgraduate Medicine, Vol. 46, No. 4, 2000, pp. 312-313.
[18] G. Escolar and M. Heras, “Clopidogrel: A Selective Inhi-bitor of Platelet ADP Receptors,” Drugs Today, Vol. 36, No. 4, 2000, p. 187.
[19] A. A. Weber, S. Reimann and K. Schr?r, “Specific Inhi-bition of ADP Induced Platelet Aggregation by Clopido-grel in Vitro,” British Journal of Pharmacology, Vol. 126, No. 2, 1999, pp. 415-420. doi:10.1038/sj.bjp.0702276
[20] K. Schr?r. “The Basic Pharmacology of Ticlopidine and Clopidogrel,” Platelets, Vol. 4, No. 5, 1993, pp. 252-261. doi:10.3109/09537109309013225
[21] P. A. Gurbel, C. M. O’Connor, C. C. Cummings and V. L. Serebruany, “Clopidogrel: The Future Choice for Pre-venting Platelet Activationduring Coronary Stenting?” Pharmacological Research, Vol. 40, No. 2, 1999, pp. 107-111. doi:10.1006/phrs.1999.0478
[22] CAPRIE Steering Committee, “A Randomized Blinded Trial of Clopidogrel versus Aspirin in Patients at Risk of Ischemic Events (CAPRIE),” Lancet, Vol. 348, No. 9038, 1996, pp. 1329-1339. doi:10.1016/S0140-6736(96)09457-3
[23] P. Lagorce, Y. Perez, J. Ortiz, J. Necciari and F. Bressolle, “Assay Method for the Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by Gas Chromatogra-phy–Mass Spectrometry,” Journal of Chromatography B: Biomedical Sciences, Vol. 720, No. 1-2, 1998, pp. 107- 117. doi:10.1016/S0378-4347(98)00452-6
[24] S. S. Singh, K. Sharma, D. Barot, P. R. Mohan and V. B. Lohray, “Estimation of Carboxylic Acid Metabolite of Clopidogrel in Wistar Rat Plasma by HPLC and Its Ap-plication to a Pharmacokinetic Study,” Journal of Chro-matography B. Analytical Technologies in the Biomedical and Life Sciences, Vol. 821, No. 2, 2005, pp. 173-180. doi:10.1016/j.jchromb.2005.05.013
[25] E. Souri, H. Jalalizadeh, A. Kebriaee-Zadeh, M. Shekarchi and A. Dalvandi, “Validated HPLC Method for De-termination of Carboxylic Acid Metabolite of Clopidogrel in Human Plasma and Its Application to a Pharmacokinetic Study,” Biomedical Chromatography, Vol. 20, 2006, pp. 1309-1314. doi:10.1002/bmc.697
[26] ICH Steering Committee, “Validation of Analytical Pro-cedures: Methodology,” 1996. http://www.pharmweb.net/pomirroor/pw9/ifpma/ichl.html
[27] U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), “Guidance for Industry: Bioanalytical Method Validation,” May 2001.
[28] A. Mitakos and I. Panderi, “Determination of the Carboxylic Acid Metabolite of Clopidogrel in Human Plasma by Liquid Chromatography–Electrospray Ionization Mass Spectrometry,” Analytica Chimica Acta, Vol. 505, No. 1, 2004, pp. 107-114. doi:10.1016/S0003-2670(03)00019-9
[29] A. Robinson, J. Hillis, C. Neal and A. C. Leary, “The Validation of a Bioanalytical Method for the Determination of Clopidogrel in Human Plasma,” Journal of Chr- omatography B, Vol. 848, No. 2, 2007, pp. 344-354. doi:10.1016/j.jchromb.2006.10.076
[30] A. Lainesse, Y. Ozalp, H. Wong and R. S. Alpan, “Bioe-quivalence Study of Clopidogrel Bisulfate Film-Coated Tablets,” Arzneimittelforshung, Vol. 54, No. 9, 2004, pp. 600-604.
[31] B. S. Shin and S. D. Yoo “Determination of Clopidogrel in Human Plasma by Liquid Chromatography/Tandem Mass Spectrometry: Application to a Clinical Pharmaco-kinetic Study,” Biomedical Chromatography, Vol. 21, No. 9, 2007, pp. 883-889. doi:10.1002/bmc.850
[32] D. Taubert, A. Kastrati, S. Harlfinger, O. Gorchakova, A. Lazar, N. Beckerath, A. Schomong and E.Schomig, “Pharmacokinetics of Clopidogrel after Administration of a High Loading Dose,” Thromb Haemostasis, Vol. 92, 2004, pp. 311-316.
[33] H. Mani, S. W. Toennes, B. Linnemann, D. A. Urbanek, J. Schwonberg, G. F. Kauert and E. Lindhoff-Last, “Deter-mination of Clopidogrel Main Metabolite in Plasma: A Useful Tool for Monitoring Therapy?” Therapeutic Drug Monitoring, Vol. 30, No. 1, 2008, pp. 84-89. doi:10.1097/FTD.0b013e31815c13fd
[34] E. Abib, L. F. Duarte, M. L. P. Vanunci, D. A. Oliveira de, S. Antonelli, et al., “Comparative Biological Availability of Clopidogrel Formulation in Healthy Volunteers After a Single Dose Administration,” Journal of Bioequivalence & Bioavailability, Vol. 2, No. 2, 2010, pp. 45-49.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.