Determination by LC‐MS/MS

The LC‐MS/MS was conducted to quantify rivaroxaban, apixaban, edoxaban and edoxaban M4 metabolite. For protein precipitation and analyte extraction, 10 µl acetonitrile:water 1:1 (v/v), 25 µl extraction buffer (MassTox TDM Series A, Chromsystems Instruments and Chemicals GmbH, Gräfelfing, Germany) and 240 µl precipitation reagent (MassTox TDM Series A, Chromsystems Instruments and Chemicals) containing the internal standards (rivaroxaban 13C6, apixaban 13C,D3 and edoxaban 13C,D2) were added to 50 µl plasma. After vortexing, the samples were centrifuged at 14 000 g and 20°C for 4 min. A 20‐µl aliquot of the supernatant was diluted with 80 µl of water:methanol 8:2 (v/v) and stored at 10°C until analysis. Calibrators and quality controls were prepared in pooled plasma (Innovative Research, Novi, MI, USA); 3 µl of the extracted samples were analysed by reversed‐phase chromatography (Cortecs UPLC C18 column, 2·1 × 75 mm, 1·7 µm; Waters Corp., Milford, MA, USA) on a triple quadrupole mass spectrometer (Xevo TQ‐S, Waters) coupled to a UPLC Acquity I‐Class system (Waters). Rivaroxaban, apixaban, edoxaban and edoxaban M4 were separated at 0·4 ml/min with a gradient using water (A) and methanol (B) acidified with 0·1% (v/v) formic acid as mobiles phases (0·0–0·5 min, 20% B; 0·5–2·5 min, 20–99% B; 2·5–3·5 min, 99% B; 3·5–3·51 min, 99–20% B; 3·51–4·5 min, 20% B). The source offset and transition parameters were optimised for each analyte. The raw data were processed with TargetLynx available in the MassLynx software (version 4.1, Waters). Edoxaban M4 metabolite was summed with edoxaban for further analysis. Rivaroxaban, apixaban and edoxaban pure substances were provided by Bayer AG, Bristol Myers Squibb Company and Daichi Sankyo Co, Ltd. (Tokyo, Japan). Clinical information and index test results were not available to the performer of the LC‐MS/MS.