Development, Validation, and Application for Simultaneous Assay of Metformin and Sitagliptin in Human Plasma by Liquid ChromatographyTandem Mass Spectrometry

Background: The objective of this study was to develop a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of metformin and sitagliptin in human plasma and to apply this method for bioequivalence study. Methods: An LC–MS/MS method was developed, validated, and applied to the quantification of metformin and sitagliptin in human plasma. Phenformin was used as an internal standard. LC–MS/MS with electrospray ionization (ESI) in positive ion mode, performed under the multiple reaction monitoring (MRM) mode, was used for analysis of the analytes. Results: The method was developed and validated with respect to selectivity and carryover, as well as intra- and inter-day accuracy and precision, according to the United States Food and Drug Administration guidance. Analytes were extracted from human plasma by a protein precipitation technique using methanol after acidification with formic acid. The mean extraction recovery for the analytes was between 70% and 99.95%, and matrix effects had only a minor influence on precision. Conclusion: The validated method was applied for a clinical bioequivalence study to evaluate the in vivo bioequivalence of a product containing 500 mg metformin hydrochloride and 50 mg sitagliptin and the Janumet XR® 50/500 mg commercial product. Fourteen healthy subjects participated in this randomized, two-period, two-treatment, open label, crossover-design study. Standard pharmacokinetic parameters were calculated to compare the test product to the commercially available Janumet XR® 50/500 mg reference product.