Determination of Coumarin-type Anticoagulants in Human Plasma by HPLC-Electrospray Ionization Tandem Mass Spectrometry with an Ion Trap Detector

Abstract

Abstract Background: Coumarin-type anticoagulants are used for the long-term treatment and prevention of thromboembolic disorders. The identification of these drugs is crucial in patients with an increased prothrombin time of unknown origin. The aim of this study was to develop a sensitive and specific method for the simultaneous determination of phenprocoumon, acenocoumarol, and warfarin in human plasma by HPLC-electrospray ionization tandem mass spectrometry. Methods: After addition of the internal standard, p-chlorowarfarin, plasma samples were extracted using Oasis® MCX solid-phase extraction cartridges. The compounds were separated on a Symmetry C18 column (Waters) with a mobile phase of acetonitrile–1 g/L formic acid (75:25 by volume) at a flow rate of 0.5 mL/min. Results: Extraction and separation of the three drugs and the internal standard were accomplished in 9 min. The overall extraction efficiency was >89% for all three compounds. The limits of detection were 1 μg/L for phenprocoumon and warfarin and 10 μg/L for acenocoumarol. Regression analysis of the calibration data revealed good correlation (r2 ≥0.995) for all compounds. Within-run accuracies for quality-control samples were ± 1% to 7% of the target concentration, with CVs <9%. Conclusions: The proposed method enables the unambiguous identification and quantification of phenprocoumon, warfarin, and acenocoumarol in both clinical and forensic specimens. This method combines a new, rapid solid-phase extraction procedure with an extremely fast chromatographic analysis, which is especially advantageous for clinical laboratories.