Validation of a method for quantitation of the clopidogrel active metabolite, clopidogrel, clopidogrel carboxylic acid, and 2-oxo-clopidogrel in feline plasma

Introduction: The clopidogrel active metabolite (CAM) is unstable and challenging to quantitate. The objective was to validate a new method for stabilization and quantitation of CAM, clopidogrel, and the inactive metabolites clopidogrel carboxylic acid and 2-oxo-clopiodgrel in feline plasma. Animals: Two healthy cats administered clopidogrel to demonstrate assay in vivo utility. Materials and methods: Stabilization of CAM was achieved by adding 2-bromo3'nnethoxyacetophenone to blood tubes to form a derivatized CAM (CAM -D). Method validation included evaluation of calibration curve linearity, accuracy, and precision; within and between assay precision and accuracy; and compound stability using spiked blank feline plasma. Analytes were measured by high perf9rmance liquid chromatography with tandem mass spectrometry. In vivo utility was demonstrated by a pharmacokinetic study of cats given a single oral dose of 18.75mg clopidogrel. Results: The 2-oxo-clopidogrel metabolite was unstable. Clopidogrel, CAM -D, and clopidogrel carboxylic acid appear stable for 1 week at room temperature and 9 months at 80 C. Standard curves showed linearity for CAM -D, clopidogrel, and dopidogrel carboxylic acid (r> 0.99). Between assay accuracy and precision was <2.6% and <7.1% for CAM -D and <= 17.9% and <11.3% for clopidogrel and clopidogrel carboxylic acid. Within assay precision for all three compounds was <= 7%. All three compounds were detected in plasma from healthy cats receiving clopidogrel. Discussion: This methodology is accurate and precise for simultaneous quantitation of CAM-D, clopidogrel, and clopidogrel carboxylic acid in feline plasma but not 2-oxo-clopidogrel. Conclusions: Validation of this assay is the first step to more fully understanding the use of clopidogrel in cats. (C) 2017 Elsevier B.V. All rights reserved.