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 (r² ≥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.

Keywords:

• Anticoagulant
• dabigatran
• β-glucuronide
• LC–MS/MS
• proton pump inhibitor
• pharmacokinetics

Acknowledgements

The authors sincerely acknowledge the support, in terms of providing necessary infrastructure and resources, from the management of the Macleods Pharmaceuticals Ltd., to carry out this work.

Ethical approval

The study was approved by Anveshhan Independent Ethics Committee– Ahmedabad, India (an independent ethics committee approved by the Drug Controller General of India (DCGI) with registration number: ECR/24/Indt/GJ/2013/RR-19).

Informed consent

Written and signed informed consent was obtained from the subjects before their inclusion in the trial.

Disclosure statement

The authors declare that there are no conflicts of interest.