Development and validation of a quantification method for direct oral anticoagulants from capillary blood using volumetric absorptive microsampling and online SPE-LC-MS

The number of prescriptions for new direct oral anticoagulants (DOACs) apixaban, edoxaban, rivaroxaban and dabigatran has increased exponentially in recent years, increasingly replacing the old gold standard, vitamin-K-antagonists. Due to their wide therapeutic range, therapeutic drug monitoring (TDM) is not required, although it has been proven that this could significantly reduce side effects. In order to develop a cost-efficient and simple method for the simultaneous detection of the DOACs and phenprocoumon, a new technology for sample preparation from capillary blood in the ambulant sector named VAMS® was integrated and an LC-MS detector with on-line solid phase extraction (SPE) applying a Turboflow HTLC Cyclone^TM 1.0x50 mm column was used. The mobile phase consisted of methanol with water (3/97 v/v) and 0.1 % ammonia solution with a flow rate of 2.5 mL/min. For the chromatographic separation, a Phenomenex LTD Kinetex 2.6 µm C18 100 Å, 100x3.0 mm column with a flow rate of 0.3 mL/min in gradient mode was utilized. The mobile phase consisted of acetonitrile, water and formic acid (A: 10:90:0.1 v/v and B: 95:05:0.1 v/v). The method was fully validated in the therapeutic range of the substances according to current guidelines. The LLOQ ranged from 3.5 µg/L for rivaroxaban to 88 µg/L for phenprocoumon and the intra-day and inter-day precision was less than 13 % and 12 %, while the accuracy was within a range of 85.7-113 % and 88.7-106 %, respectively. Samples could be stored in the Mitra® devices for at least seven days at room temperature except of dabigatran. Because the Mitras® were used, exactly 10 µL of blood could be drawn and no significant haematocrit effect was observed. A reliable, simple and cost-effective extraction and analysis LC-MS method could be developed and validated. This method is therefore applicable in ambulatory care.