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

Abstract

Objectives To validate a new method for stabilization and quantitation of the clopidogrel active metabolite (CAM), clopidogrel, and inactive clopidogrel carboxylic acid and 2-oxo-clopiodgrel. Animals Feline plasma was used for assay validation. A pilot pharmacokinetic study was conducted with 2 healthy cats. Methods CAM stabilization was achieved by adding 2-bromo-3′methoxyacetophenone to blood tubes to form a derivatized CAM (CAM-D). CAM-D was quantified using high performance liquid chromatography and tandem mass spectrometry. Validation of the methodology included evaluation of calibration curve linearity, accuracy and precision, and stability using quality control samples spiked with CAM-D, clopidogrel, clopidogrel carboxylic acid, and 2-oxo-clopidogrel. In vivo utility of this assay was evaluated by conducting a pharmacokinetic study in cats receiving a single oral dose of 18.75mg clopidogrel. Results The 2-oxo-clopidogrel metabolite was unstable. CAM-D, clopidogrel, 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 clopidogrel carboxylic acid (r>0.99). Between assay accuracy and precision was ≤2.6% and ≤2.8% 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. Conclusions This methodology is accurate and precise for simultaneous quantitation of CAM-D, clopidogrel, and clopidogrel carboxylic acid in feline plasma but unsuitable for 2-oxo-clopidogrel. Validation of this assay is the first step to more fully understanding the use of clopidogrel in cats in both a population and individual setting.

High interindividual variability in plasma clopidogrel active metabolite concentrations in healthy cats is associated with sex and cytochrome P450 2C genetic polymorphism

“…Because pharmacokinetic parameters will vary between individuals, sampling at 2 hr may reflect C max for certain individuals but not others. This variation has been shown by our group in a previously reported pilot pharmacokinetic study (Lyngby et al., ).…”

“…A Synergi Phenomenex Polar‐RP 2.0 × 150 mm column was used for separation at a column temperature of 30°C. Specific details regarding the instrument settings and assay performance, including accuracy and precision, are provided elsewhere (Lyngby et al., ).…”

“…The clopidogrel active metabolite covalently binds to the platelet P2Y 12 receptor resulting in irreversible inhibition of ADP‐induced platelet activation and aggregation. The clopidogrel active metabolite is labile but can be stabilized and quantitated by adding the compound 2‐bromo‐3′methoxyacetophenone (BMAP) to blood tubes to form a derivatized CAM (CAM‐D) (Figure ) (Karazniewicz‐Lada et al., ; Lyngby, Court, & Lee, ; Peer et al., ).…”

Influence of CYP2C19 Phenotype on the Effect of Clopidogrel in Patients Undergoing a Percutaneous Neurointervention Procedure

“…The main limitation of our study is the difficulty of measuring the pharmacokinetic parameters of the clopidogrel active metabolite; therefore, we could not objectively quantify the amount of drug metabolized, and thus correlate it with the aggregation value and subsequent events. A previous study demonstrated that CYP2C19 *2 allele has an influence on clopidogrel concentrations, and thus on exposure to its metabolite .…”

Population variability in animal health: Influence on dose–exposure–response relationships: Part I: Drug metabolism and transporter systems

“…High variability has also been reported in the effects of clopidogrel on platelet function in different cats (Teuber & Mischke, ). Unpublished data using a newly developed assay (Lyngby, Court, & Lee, ) were presented at the meeting by a coauthor (MHC) showing over 10‐fold variation in active metabolite concentrations among 19 cats given clopidogrel. Consequently, a polymorphism in the feline CYP2C ortholog could explain this variability, and variant genotyping may ultimately be used to identify cats that are resistant to clopidogrel treatment.…”