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 |
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Authors: | Silvestro Luigi Gheorghe Mihaela Iordachescu Adriana Ciuca Valentin Tudoroniu Ariana Rizea Savu Simona Tarcomnicu Isabela |
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Institution: | (1) 3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany;(2) Pharma Serv Int’l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania;(3) Faculty of Chemistry, Department of Analytical Chemistry, University of Bucharest, 90 Panduri Av., 050663 Bucharest, Romania |
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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. |
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