Rapid LC--ESI-MS--MS Method for the Simultaneous Determination of Clopidogrel and its Carboxylic Acid Metabolite in Human Plasma

Abstract

A high throughput liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS-MS) method is developed for the simultaneous estimation of clopidogrel (SR25990C) and its carboxylic acid metabolite (SR26334) in human plasma using glimepiride as internal standard. The extraction of SR25990C, its metabolite, and IS from the plasma (0.3 mL) involves treatment with phosphoric acid followed by solid-phase extraction (SPE). Sample preparation by this method yields clean extracts with quantitative and consistent mean recoveries of 98.05%, 85.45%, and 105.72% for SR25990C, SR26334, and IS, respectively. The SPE eluate without drying and reconstitution is analyzed by LC-MS-MS, operating in the positive ion and selective reaction monitoring mode. The injection volume is 2 microL with a total chromatographic run time of 5.0 min. The method response is linear over the dynamic range of 0.25 to 25.0 ng/mL for SR25990C and 50.0 to 6000.0 ng/mL for SR26334, with correlation coefficients of r > or = 0.9989 and 0.9984, respectively. The method is validated to demonstrate its specificity, linearity, accuracy, precision, recovery, matrix effect, dilution integrity, and stability studies. It is applied to study the bioavailability of 75 mg clopidogrel mesylate tablets in 16 human subjects with satisfactory results.

A sensitive and rapid ultra HPLC–MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma

“…Consequently, few studies such as these have quantified the active metabolite in order to determine differences in exposure levels between patients with genotypes [5,7]. Many previous studies have reported analytical methods quantifying either parent clopidogrel [10–13] or clopidogrel and its inactive metabolites (resulting from carboxyesterase activity) in an effort to measure clopidogrel active metabolite (CAM) indirectly [14–18]. Another group attempted semiquantitation of underivatized CAM, using clopidogrel to establish the calibration curve [19], but this provided only an estimate of active metabolite plasma concentrations.…”

The Personalization of Clopidogrel Antiplatelet Therapy: The Role of Integrative Pharmacogenetics and Pharmacometabolomics

“…Several studies had evaluated different pharmacokinetics (PK) methods to determine the level of the parent compound clopidogrel and its metabolites as an indicator of clopidogrel platelets inhibition [5, 99, 100]. It was found that the level of clopidogrel active metabolite is correlated with clopidogrel platelets inhibition [86, 101], and the PK of clopidogrel could be predicted by the CYP2C19 genotype [102].…”

“…The active thiol metabolite of clopidogrel is not stable which requires adding stabilizing agent within 30 seconds of blood sampling [101]. Some studies used indirect quantification of the active metabolite by quantifying the parent compound and the inactive metabolite [99, 103]. This means an accurate quantification of the active thiol metabolite in blood is difficult.…”

HPLC‐PDA‐ORD Bioassay of S‐(+) and R‐(−) Clopidogrel on Rat Dried Blood Spots

“…Several methods, including high‐performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS), and ultra performance liquid chromatography (UPLC)‐MS were reported to analyze clopidogrel in biological matrices. Determination of the carboxylic acid metabolite of clopidogrel in human, rat plasma and serum by liquid–liquid extraction followed by HPLC have been reported .…”

“…Determination of the carboxylic acid metabolite of clopidogrel in human, rat plasma and serum by liquid–liquid extraction followed by HPLC have been reported . Simultaneous determination of clopidogrel, its carboxylic acid, and thiol metabolites in clinical samples by liquid chromatography tandem mass spectroscopy (LC‐MS/MS) were reported . UPLC‐MS/MS was also use to determine clopidogrel in dog plasma .…”