A liquid chromatography-tandem mass spectrometry method for the determination of apixaban in human plasma and its application to pharmacokinetics studies in the Indian population.

Apixaban is a novel oral anticoagulant intended to treat and prevent blood clots and to prevent strokes in patients with nonvalvular atrial fibrillation. The development and validation of a fast, selective, accurate, and precise method using high-performance liquid chromatography tandem mass spectrometry is described for the estimation of apixaban in human plasma, with apixaban 13CD3 as an internal standard (IS). Using a reverse phase Gemini C18 column (50 mm × 4.6 mm, 3 μm) and a mixture of acetonitrile (2 mM) and ammonium formate buffer (50 : 50 v/v) as the mobile phase, chromatographic separation was achieved following extraction via a solid-phase extraction process. To track multiple reaction monitoring transitions set at 460/443 (m/z) and 464/447 (m/z) for apixaban and apixaban 13CD3, respectively, liquid chromatography coupled with triple quadrupole mass spectrometry was employed. A concentration linearity range between 1.01 and 280.00 ng mL−1 was validated with regression ≥0.99, and the method was successfully applied to apixaban pharmacokinetics analysis. At a flow rate of 1.0 mL min−1, the run time was around 1.8 min, which is short. With an extraction recovery of >73% for both apixaban and apixaban 13CD3, the method was sensitive, with a limit of quantitation of 1.01 ng mL−1. The inter-day/between-run precision ranged from 1.21% to 3.21%, while the accuracy ranged from 96.5% to 102%. For pharmacokinetics analysis, the validated method was applied. The percentage difference between findings from samples that were reanalyzed and samples that were initially analyzed was within ±20%. With high-quality assay specificity and accuracy in relation to apixaban analysis in human plasma under the experimental conditions used, the method provided is accurate.