Masstox tdm series

Liquid chromatography-tandem mass spectrometry was used for the quantification of rivaroxaban, apixaban, edoxaban, and M4 metabolite of edoxaban. For protein precipitation and analyte extraction, plasma acetonitrile:water 1:1 (v/v), extraction buffer (MassTox TDM Series A, Chromsystems, Gräfelfing, Germany), and precipitation reagent (MassTox TDM Series, Chromsystems, Gräfelfing, Germany) containing the isotope labeled internal standards (13C6 rivaroxaban, 13CD3 apixaban, 13CD2 edoxaban; provided by the manufacturers) were added to the plasma. Afterward, the samples were vortexed and centrifuged at 14,000 rcf and 20°C for 4 min. The supernatant was diluted with water:methanol 8:2 (v/v) and stored at 10°C until analysis. The calibrators and QCs were prepared in pooled plasma (Innovative Research, Novi, MI, United States). The extracted samples were analyzed using reversed-phase chromatography (Cortecs UPLC C18 column, 2.1 × 75 mm, 1.7 μm, Waters) on a triple quadrupole mass spectrometer (Xevo TQ-S, Waters, Milford, CT, United States) coupled to a UPLC Acquity I-Class system (Waters, Milford, CT, United States). Edoxaban M4 concentration was summed up with the edoxaban for further analysis.

Rivaroxaban, apixaban, edoxaban, and edoxaban M4 were quantified by LC-MS/MS as previously described (20 (link)). Briefly, protein precipitation and analyte extraction were performed by adding to the plasma acetonitrile:water 1:1 (v/v), extraction buffer (MassTox TDM Series A, Chromsystems, Gräfelfing, Germany), and precipitation reagent (MassTox TDM Series, Chromsystems, Gräfelfing, Germany) containing the isotope labeled internal standards (13C6 rivaroxaban, 13CD3 apixaban, 13CD2 edoxaban) The samples were vortexed and then centrifuged at 14000 rcf and 20°C for 4 min. The supernatant was diluted with water: methanol 8:2 (v/v) and stored at 10°C until analysis. Calibrators and QCs were prepared in pooled plasma. Then, the extracted samples were analyzed by reversed-phase chromatography on a triple quadrupole mass spectrometer (Xevo TQ-S, Waters, Milford, USA) coupled to a UPLC Acquity I-Class system (Waters, Milford, USA). Edoxaban M4 concentration was summed up with edoxaban concentration for further analysis.

Rivaroxaban, Edoxaban, Edoxaban M4, and Apixaban plasma concentrations were determined using LC-MS/MS, as previously described [27 (link)]. The following substances were added to the plasma samples for protein precipitation and extraction: extraction buffer (MassTox TDM Series A, Chromsystems, Gräfelfing, Germany), acetonitrile:water 1:1 (v/v), and precipitation reagent containing the isotope labeled standards 13C6 rivaroxaban, 13CD3 apixaban, and 13CD2 edoxaban (MassTox TDM Series, Chromsystems, Gräfelfing, Germany). The samples were swirled and centrifuged, and water:methanol 8:2 (v/v) was added to the supernatant. Reversed-phase chromatography was used on a triple quadrupole mass spectrometer (Xevo TQ-S, Waters, Milford, CT, USA) coupled to a UPLC Acquity I-Class system (Waters, Milford, CT, USA) for analysis. The Edoxaban M4 and Edoxaban concentrations were summed up for the analysis.