Simultaneous quantification of acetylsalicylic acid, clopidogrel, ticagrelor and their major metabolites in human plasma by liquid chromatography-tandem mass spectrometry

In recent years, there has been a critical need for therapeutic drug monitoring (TDM) of antiplatelet agents. To address this, we developed a sensitive, rapid, and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method as a unified assay for the simultaneous quantification of aspirin (ASA), salicylic acid (SA), clopidogrel (CLP), its carboxylic acid metabolite (CLPM), ticagrelor (TGL), and its active metabolite AR-C124910XX for the first time. Plasma samples were pretreated by protein precipitation and separated on an ACQUITY UPLC BEH C18 column (2.1 × 50 mm, 1.7 μm) with a total run time of 4 min. Mass spectrometry was performed in polarity-switching mode, and quantification was achieved using multiple reaction monitoring (MRM) with stable isotope-labeled internal standards (SA-d6, CLPM-d4, TGL-d7) for normalization. Method validation demonstrated excellent linearity over the range of 0.5-2000 ng/mL (R² ≥ 0.9938), with extraction recoveries of 98.2 %-107 % and negligible matrix effects. Stability testing indicated that all analytes were generally stable under clinically relevant conditions; however, ASA underwent rapid esterase-mediated hydrolysis at room temperature, forming SA as the major degradation product, whereas it remained relatively stable at 2-8 °C and was stable for at least 15 days at -80 °C. The method was successfully applied to plasma samples from 61 cardiovascular patients, confirming its utility for evaluating medication adherence as well as inter- and intra-individual variability in drug exposure. In conclusion, the developed LC-MS/MS method provides high sensitivity, high throughput, and robust performance, offering a powerful analytical tool to support individualized antiplatelet therapy.

Acetylsalicylic acid; Clopidogrel; LC–MS/MS; Pharmacokinetics; Therapeutic drug monitoring; Ticagrelor.