A sensitive and simple liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous quantification of ticagrelor and its active metabolite, AR-C124910XX from 50 μL human plasma using tolbutamide as an internal standard as per regulatory guidelines. Analytes in plasma were extracted by simple protein precipitation using acetonitrile, followed by chromatographic separation with an Acclaim™ RSLC 120 C18 column (2.2 μm, 2.1 × 100 mm) and a gradient acetonitrile-water mobile phase containing 0.1% formic acid within 8 min. Mass spectrometric detection and quantitation were conducted by selected reaction-monitoring on a negative electrospray ionization mode with the following transitions: m/z 521.11 → 361.10, 477.03 → 361.10, and 269.00 → 169.60 for ticagrelor, AR-C124910XX, and tolbutamide, respectively. The lower limit of quantifications was 0.2 ng/mL with linear ranges of 0.2−2,500 ng/mL (r2 ≥ 0.9949) for both analytes. All validation data, including selectivity, cross-talk, precision, accuracy, matrix effect, recovery, dilution integrity, stability, and incurred sample reanalysis, were well within acceptable limits. This assay method was validated using K2-EDTA as the specific anticoagulant. Also, the anticoagulant effect was tested by lithium heparin, sodium heparin, and K3-EDTA. No relevant anticoagulant effect was observed. This validated method was effectively used in the determination of ticagrelor and its active metabolite, AR-C124910XX, in plasma samples from patients with myocardial infarction.
Bibliographical noteFunding Information:
This research was supported by the Basic Research Laboratory (BRL) Program (2015R1A4A1042350) and the Basic Science Research Program (2017R1A2B4008761) through the National Research Foundation of Korea funded by the Korean government.
All Science Journal Classification (ASJC) codes
- Pharmacology (medical)