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

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 D-4. The extraction of analyte arid IS was accomplished by the solidphase extraction technique. Chromatographic separations were achieved using Peerless basic C8 (150 x 4.6) mm, 5 mu column eluted at a flow rate of 1 mL/min with mobile phase Acetonitrile: 5mM 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 D-4 at around 1.2 min. The multiple reaction monitoring transitions (Q(1)/Q(3)) were set at 472/289, 172 (m/z) for dabigatran, arid 476/293 (m/z) for dabigatran D-4. 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.