Development and validation of an HPLC–MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion

Abstract

A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80-120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C(max) of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.

Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man and Mice of Clopidogrel Acyl Glucuronide

“…The present studies represent a follow-up to previous work from which we reported the existence of CAG and described its in vitro back-conversion to clopidogrel by transesterification (Silvestro et al, 2011). The main questions to clarify now are, "Can this happen by any means in vivo also?"…”

“…It was shown that in specific conditions it converts to parent clopidogrel by trans-esterification (Silvestro et al, 2011), a reaction sometimes occurring also during metabolic processes (Boyer and Petersen, 1992;Knights et al, 2000;Celli et al, 2007;Fujino et al, 2014).…”

Clarification of the Mechanism of Clopidogrel-Mediated Drug-Drug Interaction in a Clinical Cassette Small-dose Study and Its Prediction Based on In Vitro Information

“…The plasma concentrations of all compounds were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a Shimadzu LCMS-8050 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). All sample pretreatment procedures were conducted on ice using an aluminum cooling block to prevent the backconversion of clopidogrel acyl-b-glucuronide to clopidogrel carboxylic acid or clopidogrel (Silvestro et al, 2011).…”

Reversible Inhibition of Human Carboxylesterases by Acyl Glucuronides

“…This IS/quench solution contained 10% acetic acid for all acyl glucuronide solutions except for clopidogrel-b-D-glucuronide, which contained only 0.5% acetic acid. The IS/quench solution for clopidogrel-b-D-glucuronide was used to keep the samples near a pH of 7 since this acyl glucuronide was shown to be less stable at a more acidic or basic pH (Silvestro et al, 2011). Samples were analyzed using LC/TOFMS (liquid chromatography interfaced with time-offlight mass spectrometry).…”