HPLC–MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples

Karaźniewicz−Łada, Marta; Danielak, Dorota; Tężyk, Artur; Żaba, Czesław; Tuffal, Gilles; Główka, Franciszek

doi:10.1016/j.jchromb.2012.11.005

Cited by 48 publications

(47 citation statements)

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“…After oral administration of clopidogrel to humans, only H3 and H4 could be detected, and had similar concentrations in plasma, whereas H1 and H2 were not detected (Tuffal et al, 2011;Kara zniewicz-Łada et al, 2012). Therefore, H4 is the only isomer responsible for the antiplatelet activity dx.doi.org/10.1124/dmd.115.064949. s This article has supplemental material available at dmd.aspetjournals.org.…”

Section: Introductionmentioning

confidence: 99%

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

et al. 2015

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Clopidogrel, a thienopyridine antiplatelet prodrug, is metabolized by oxidation to 2-oxo-clopidogrel, followed by conversion to its pharmacologically active thiol metabolite. After oral administration of clopidogrel to humans, two thiol isomers (H3 and H4) are observed in plasma, with similar concentrations, and only H4 is active in humans. In this work, the mechanism of stereoselectivity in the formation and S-methylation of H3 and H4 was investigated in vitro. The two diastereomers of 2-oxo-clopidogrel were epimerized rapidly at physiologic pH. The intrinsic clearance (CL int ) for H3 formation from 2-oxo-clopidogrel in human liver microsomes (HLMs) was 3.1-fold higher than that for H4 formation, indicating stereoselective metabolism. Kinetic studies using expressed enzymes demonstrated that the contributions of CYP2B6, CYP2C19, and CYP3A4 to the formation of H4 from 2-oxo-clopidogrel were 18.5%, 26.1%, and 53.5%, respectively. The CL int ratios of H3 formation to H4 formation from 2-oxo-clopidogrel by CYP2B6, CYP2C19, and CYP3A4 were 2.2, 1.0, and 1.7, respectively. In HLMs, H3 and H4 were further S-methylated, and the S-methylation was inhibited by 2,3-dichloromethyl benzylamine, indicating the involvement of thiol S-methyltransferase. The CL int value for the S-methylation of H3 in HLMs was 98.1-fold higher than that for H4. The stereoselective formation of H3 from 2-oxo-clopidogrel and the stereoselective S-methylation of H3 may lead to the similar exposure levels of H3 and H4 previously reported in humans. The epimerization of 2-oxoclopidogrel and the variations of thiol S-methyltransferase may affect the exposure to H4 in humans.

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Section: Introductionmentioning

confidence: 99%

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

et al. 2015

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“…The C max value of the 75 mg clopidogrel tablet was 2.1 ng/mL; therefore, the LLOQ should not exceed 105 pg/mL. This method did not meet the requirement; however, the LLOQ obtained was lower than that in the study by Karaźniewicz-Ładaz [9].…”

Section: Lloq Measurementmentioning

confidence: 68%

Analytical Validation of Clopidogrel in Human Plasma Through Ultrahighperformance Liquid Chromatography-Tandem Mass Spectrometry

Harahap

Maysyarah

2017

Int J App Pharm

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Objective: This study developed a sensitive, selective, and valid method for analyzing clopidogrel in human plasma using liquid chromatographytandem mass spectrometry (LC-MS/MS). Methods:The chromatography separation was performed on the waters ACQUITY UPLC Class BEH C 18 1.7 µm (2.1 × 100 mm) column, consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (30-70) as the mobile phase, and an isocratic elution with a flow rate of 0.2 mL/minutes. The mass detection was performed using a Waters Xevo TQD equipped with positive electrospray ionization in the multiple reaction monitoring modes. The clopidogrel was detected at m/z 322.086>212.097 and irbesartan as an internal standard at m/z 429.233>207.131. The sample was prepared with protein precipitation method using acetonitrile, vortex mixed for 10 minutes, and centrifuged at 13,000 rpm for 20 minutes.Results: This method showed a linear result at the concentration range of 0.2-10 ng/ml with r≥0.9997. Conclusions:The developed method provides sensitivity, linearity, precision, and accuracy and is suitable for analysis of clopidogrel in human plasma using LC-MS/MS.

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“…It is primarily metabolized to the inactive clopidogrel acid, generated by ester hydrolysis, and to the pharmacologically active metabolites (CAM) via the inactive 2-oxoclopidogrel by a two-step cytochrome P450 oxidation process [1][2][3][4]. In the ring opening of 2-oxoclopidogrel to form CAM, two different stereochemical processes occur to form a mixture of four diastereomers and are referred to as H1 to H4 (Figure 1) [2,5]. The biological activity of H1 to H4 has been evaluated and H4 was reported as a major active diastereomer [2,6].…”

mentioning

confidence: 99%

“…Karaźniewicz-Łada et al [5] reported an HPLC-MS/MS method for the simultaneous determination of clopidogrel, clopidogrel acid and derivatized H3 and H4 (MP-H3, MP-H4) with an LLOQ of 0.25 ng/ml for MP-H3, MP-H4 and clopidogrel. In this method, MP-2 and MP-H3 were not separated.…”

mentioning

confidence: 99%

“…In this method, MP-2 and MP-H3 were not separated. It is desirable to have very low LLOQ for both clopidogrel and H4 due to the low C max and short halflife for both compounds [5,14]. In this study, we present method development data for a sensitive and fast UHPLC-MS/MS method to accurately quantify the clopidogrel, clopidogrel acid and CAM H4 in human plasma samples.…”

mentioning

confidence: 99%

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Development of A Sensitive and Fast UHPLC–MS/MS Method for Determination of Clopidogrel, Clopidogrel Acid and Clopidogrel Active Metabolite H4 in Human Plasma

et al. 2015

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HPLC–MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples

Cited by 48 publications

References 20 publications

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

Human Liver Cytochrome P450 Enzymes and Microsomal Thiol Methyltransferase Are Involved in the Stereoselective Formation and Methylation of the Pharmacologically Active Metabolite of Clopidogrel

Analytical Validation of Clopidogrel in Human Plasma Through Ultrahighperformance Liquid Chromatography-Tandem Mass Spectrometry

Development of A Sensitive and Fast UHPLC–MS/MS Method for Determination of Clopidogrel, Clopidogrel Acid and Clopidogrel Active Metabolite H4 in Human Plasma

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