The Development of a Liquid Chromatography High-Resolution Mass Spectrometric Method for Apixaban Quantification in Dried Plasma Spots in Parallel Reaction Monitoring Mode

Abstract

This work aimed at developing and validating a rapid, sensitive, and robust method of liquid chromatography with high-resolution mass spectrometry (LC–HRMS) in parallel reaction monitoring (PRM) mode for apixaban quantification in dried plasma spots (DPSs) with a simple extraction procedure. A 25 µL sample of human plasma was placed onto Whatman 903 Protein Saver Cards and allowed to dry; 3.2 mm diameter disks were cut out from DPSs using a puncher, and 100 µL of a working internal standard solution was added to each sample. After this, they were vortexed on a shaker for 15 min at 800 rpm and 40 °C and quick centrifugation (10,000× g, 10 s), and then the extracts were transferred into a 300 µL vial for LC–HRMS. Data were acquired in PRM mode via detection of all target product ions with 10 ppm tolerance. Total analysis time was 5 min. The LC–HRMS method was validated for the 10–400 ng/mL range with R2 > 0.99. Within this range, intra- and interday variability of precision and accuracy was <10%, and recovery was 69.7–85.1%. Apixaban was stable after brief storage at room temperature, and at 4 °C for up to a month. The method development and validation results proved that this LC–HRMS assay of apixaban in DPSs is selective and robust.