Liquid chromatography-tandem mass spectrometry simultaneous determination of repaglinide and metformin in human plasma and its application to bioequivalence study

A simple, sensitive, selective, and reproducible liquid chromatography-tandem mass spectrometric method was developed for the simultaneous determination of repaglinide and metformin in human plasma using d₅-repaglinide and d₆-metformin as internal standards (ISs).  After a simple protein precipitation using acetonitrile as the precipitation solvent, both analytes and ISs were separated on a Venusil ASB C₁₈ (150 mm × 4.6 mm, 5 μm) via gradient elution using acetonitrile − 10 mmol·L⁻¹ ammonium acetate as the mobile phase.  A chromatographic total run time of 7.5 min was achieved.  Mass spectrometric detection was conducted with atmospheric pressure chemical ionization under positive-ion and multiple-reaction monitoring modes.  The method was linear over the 0.2 to 60.0 ng·mL⁻¹ concentration range for repaglinide and over the 4 to 1 000 ng·mL⁻¹ range for metformin.  For both analytes, the intra- and inter-accuracies and precisions were within the ±15% acceptable limit across all concentrations.  The validated method was successfully applied to a clinical bioequivalence study.