A Chromatographic Approach for Simultaneous Quantification of Multiple Anticoagulants in Human Plasma Samples

Anticoagulants are essential drugs for preventing and treating thromboembolic conditions by inhibiting blood clot formation. Warfarin, a vitamin K antagonist, is among the most prescribed agents but presents limitations, including a narrow therapeutic window and a high risk of adverse events, requiring strict monitoring to ensure both safety and therapeutic efficacy. In contrast, direct oral anticoagulants such as dabigatran, rivaroxaban, and apixaban offer improved safety profiles and generally do not require routine monitoring. Nevertheless, monitoring is required in special clinical situations, including emergencies, renal dysfunction, or for high-risk patients. In this context, the present study aimed to develop an HPLC method with UV/Vis detection for the simultaneous quantification of four anticoagulant agents (warfarin, dabigatran, rivaroxaban, and apixaban) in human plasma. Efficient separation of the four target analytes involved the evaluation of several parameters, including column type (Kinetex core-shell C18, Chromolith RP-18e, and Chromolith Phenyl), elution conditions, and injection volume (10-50 µL). The best separation was achieved within 12 min using a monolithic phenyl column and gradient elution, at a flow rate of 2 mL/min. The method was validated and has shown to be selective, linear in the range of 0.50-5.0 µg/mL for dabigatran and warfarin and 0.25-5.0 µg/mL for rivaroxaban and apixaban, accurate (85.7%-115.0%), and precise (CV ≤ 9.4%) for both intra- and inter-day assays. LOD and LOQ in plasma were ≤ 0.2 and ≤ 0.5 µg/mL, respectively. Stability testing demonstrated that all analytes remained stable at room temperature for 24 h, with only dabigatran showing reduced stability after three freeze-thaw cycles. The proposed method enabled rapid and simultaneous quantification of four clinically relevant anticoagulants in plasma samples.