Liquid chromatography-tandem mass spectrometry method for determination of rivaroxaban in human plasma and its application to a pharmacokinetic study.

Abstract

A high-performance liquid chromatography tandem mass spectrometric method for the determination of Rivaroxaban in human plasma has been developed and validated using Rivaroxaban D4 as an internal standard. The extraction of analyte and internal standard was accomplished by solid phase extraction technique. The method has been validated over a concentration range of 5.96-801 ng/mL. Chromatographic separations were achieved using Gemini C18, 150 mm × 4.6 mm, 5 µm, column eluted at flow rate of 1.5 mL/min with mobile phase (acetonitrile: ammonium acetate buffer (80:20 v/v)). The overall run time of method was about 1.8 min with elution times of Rivaroxaban and its internal standard Rivaroxaban D4 at around 1.18 min. The multiple reaction monitoring transitions were set at 436/145 (m/z) and 440/145 (m/z) for Rivaroxaban and Rivaroxaban D4, respectively. The calibration curves were linear (r2 ≥ 0.99) over the range of 5.96-801 ng/mL with lower limit of quantitation validated at 5.96 ng/mL. Extraction recoveries were >88% for both rivaroxaban and its stable labeled internal standard rivaroxaban D4. The inter-day/between run precisions were ranged from 1.08% to 3.75%, while accuracy ranged from 96.3% to 102%. The presented method was used in pharmacokinetic study in healthy volunteers. Results of incurred sample reanalysis were within the acceptance range of ±20% of original value, for 98.3% of samples reanalyzed. This indicated good assay precision of target analytes in their real matrix at the employed experimental conditions. The applicability of the assay for the determination of the pharmacokinetic parameters was demonstrated.