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Clopidogrel high on treatment platelets reactivity (HTPR) has burdened achieving optimum therapeutic outcome. Although there are known genetic and non-genetic factors associated with clopidogrel HTPR, which explain in part clopidogrel HTPR, yet, great portion remains unknown, often hindering personalizing antiplatelet therapy. Nuclear magnetic resonance (1H NMR) pharmacometabolomics analysis is useful technique to phenotype drug response. We investigated using 1H NMR analysis to phenotype clopidogrel HTPR in urine. Urine samples were collected from 71 coronary artery disease (CAD) patients who were planned for interventional angiographic procedure prior to taking 600mg clopidogrel loading dose (LD) and 6h post LD. Patients' platelets function testing was assessed with the VerifyNow® P2Y12 assay at 6h after LD. Urine samples were analysed using 1H NMR. Multivariate statistical analysis was used to identify metabolites associated with clopidogrel HTPR. In pre-dose samples, 16 metabolites were associated with clopidogrel HTPR. However, 18 metabolites were associated with clopidogrel HTPR in post-dose samples. The pathway analysis of the identified biomarkers reflected that multifactorial conditions are associated with clopidogrel HTPR. It also revealed the implicated role of gut microbiota in clopidogrel HTPR. Pharmacometabolomics not only discovered novel biomarkers of clopidogrel HTPR but also revealed implicated pathways and conditions.

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A simple and sensitive spectrofluorimetric method has been developed for the determination of clopidogrel sulfate in pharmaceutical formulation and human urine/serum. It is based on the formation of ion-pair complex between clopidogrel sulfate and alizarin red in 0.3 mol x L(-1) hydrochloric acid solution. The ion pair complex was extracted in dichloromethane and the fluorescence intensity was measured at 550 nm after excitation at 428 nm. The various factors influencing ion-pair complex formation and fluorescence determination were discussed. Under the optimized conditions, the fluorescence value showed a good linear relationship with the clopidogrel sulfate concentration ranging from 1.0 to 11.0 µg x mL(-1). The equation of calibration curve was F = 53.32 + 35.01c (µg x mL(-1)), r = 0.994, and the detection limit was found to be 0.11 µg x mL(-1). No interference was observed from common co-existing substances or pharmaceutical excipient. The determination recoveries of clopidogrel sulfate in pharmaceutical formulation and human urine/serum samples were 90.6%-99.3%, 104.6%-109.3%, 96.3%-105.0%, respectively. The method was successfully applied to detect clopidogrel sulfate in clopidogrel sulfate tablet and human urine/ serum. The obtained results of clopidogrel sulfate tablet were in good agreement with the results of HPLC. Therefore, it is concluded that the proposed method is simple, sensitive and rapid for the determination of clopidogrel sulfate in real samples.

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Fast and reproducible Capillary Zone Electrophoresis (CZE) method for the quantification of (+)-S clopidogrel carboxylic acid metabolite in human fluids was elaborated for the first time. Optimal buffer and CZE conditions were established to obtain the complete separation of clopidogrel, its metabolite and piroxicam (internal standard), during one analytical run. Finally, resolution of the analytes was obtained in an uncoated silica capillary filled with a phosphate buffer of pH 2.5. The analytes were isolated from plasma and urine samples using solid phase extraction (SPE). Validation of the CZE method was carried out. The calibration curve of clopidogrel was linear in the range of 0.5-10.0mg/L in plasma and urine, whereas for (+)-S carboxylic acid metabolite linearity was confirmed in the range of 0.25-20.0mg/L in plasma and 0.25-10.0mg/L in urine. Intra- and inter-day precision and accuracy were repeatable. LOD and LOQ were also estimated. SPE recovery of the analytes from plasma and urine was comparable and greater than 80%. The validated method was successfully applied in pharmacokinetic investigations of (+)-S carboxylic acid metabolite of clopidogrel following the oral administration of clopidogrel to patients prior to percutaneous coronary intervention.

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We have identified several novel metabolites of ticlopidine, a well known antiplatelet agent and have revealed its metabolic route in rats. The main biliary metabolite of ticlopidine was characterized as a glutathione (GSH) conjugate of ticlopidine S-oxide, in which conjugation had occurred at carbon 7a in the thienopyridine moiety. Quantitative analysis revealed that 29% of the dose was subjected to the formation of reactive intermediates followed by conjugation with GSH after oral administration of ticlopidine (22 mg/kg) to rats. In vitro incubation of ticlopidine with rat liver 9000 g supernatant fraction (S9) fractions led to the formation of multiple metabolites, including 2-oxo-ticlopidine, the precursor for the pharmacologically active ticlopidine metabolite, [1-(2-chlorobenzyl)-4-mercaptopiperidin-(3Z)-ylidene] acetic acid. A novel thiophene ring-opened metabolite with a thioketone group and a carboxylic acid moiety has also been detected after incubation of 2-oxo-ticlopidine with rat liver microsomes or upon incubation of ticlopidine with rat liver S9 fractions.

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