Liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for determination of free and total dabigatran in human plasma and its application to a pharmacokinetic study

Abstract

A high-performance liquid chromatography-tandem mass spectrometric method for the determination of free and total dabigatran in human plasma has been developed and validated using a stable labeled internal standard (IS) as dabigatran D4. The extraction of analyte and IS was accomplished by the solid-phase extraction technique. Chromatographic separations were achieved using Peerless basic C8 (150 × 4.6) mm, 5 µ column eluted at a flow rate of 1 mL/min with mobile phase Acetonitrile: 5 mM ammonium formate: Methanol and 0.2% formic acid (30:20:50, v/v/v). The run time of the method was about 2.5 min with elution times of dabigatran and dabigatran D4 at around 1.2 min. The multiple reaction monitoring transitions (Q1/Q3) were set at 472/289, 172 (m/z) for dabigatran, and 476/293 (m/z) for dabigatran D4. The calibration curves were linear (r 2 ≥0.99) over the range of 1.04–406.49 ng/mL. The presented method was successfully employed in the analysis of pharmacokinetic studies with the added advantage of demonstrating the effect of co-administration of dabigatran with the proton pump inhibitor pantoprazole on bioavailability and pharmacokinetic characteristics. Re-analysis of incurred sample resulted in >98% compliance indicating good assay precision of target analytes.