Regulatory recommendations for clopidogrel: Focus on validation of a reliable bioanalytical method and its application to a bioequivalence study

Abstract

A rapid, simple, and sensitive liquid chromatography tandem mass spectrometric (LC–MS/MS) method for the determination of clopidogrel in human plasma was developed and validated using clopidogrel-d4 as the internal standard (IS). The analyte and the IS were extracted from 500 µl aliquots of human plasma via solid phase extraction. The precursor to product ion transitions monitored for clopidogrel and IS were m/z 322.2 → 212.0 and 326.2 → 216.0, respectively. The method was fully validated for sensitivity, accuracy and precision, linearity, recovery, matrix effect, dilution integrity, and stability. The Linearity range was 20.4–10,772.6 pg/mL with mean correlation coefficient (r) ≥ 0.9977. The back conversion was also evaluated during method validation as per EMA recommendation. Results proved that clopidogrel was accurately and reliably estimated by the method without any evidence of back conversion of clopidogrel acid. The method was successfully applied to a bioequivalence study of 75 mg clopidogrel bisulfate tablet formulation in 32 healthy male volunteers under fasting conditions. The ratios of least-squares means (with 90% confidence intervals) for the pharmacokinetic parameters C_max, AUC_0–t and AUC_0–α were 107.98% (94.52–123.35%), 100.25% (91.28–110.09%), and 100.49% (91.37–110.51%), respectively.

Keywords::

• Clopidogrel
• clopidogrel acid
• back conversion
• method validation
• bioequivalence

Acknowledgements

This study was conducted by Ranbaxy Research Laboratories, India. The authors wish to thank the clinical site staff, subjects, and formulation department of Ranbaxy for the enormous support and contributions received.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.