Quantification Of Repaglinide And Metformin In Human Plasma And Its Application In A Bioequivalence Study

Background Repaglinide is a fast-acting prandial oral hypoglycemic agent for patients with non-insulin-dependent diabetes mellitus (NIDDM).This substance helps control blood glucose levels by promoting insulin release from pancreatic β-cells. Metformin is an orally administered antihyperglycemic drug widely used in NIDDM treatment. This drug improves peripheral insulin sensitivity, inhibits hepatic gluconeogenesis, and reduces hepatic glucose production in patients with NIDDM. To date, no liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of repaglinide and metformin in human plasma has been reported.Objective A sensitive and specific liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method was to be developed for the simultaneous determination of repaglinide or metformin in human plasma. The method should be applied to the bioequivalence study of repaglinide or metformin.Method d5-Repaglinide and d6-metformin were used 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(150×4.6mm,5μm) via gradient elution using acetonitrile-10mmol/L ammonium acetate as the mobile phase. A chromatographic total run time of7.5min was achieved.Detection was performed by tandem mass spectrometry using an atmospheric pressure chemical ionization (APCI) source in the positive ion mode, operating in the multiple reaction monitoring (MRM) of the transitions of m/z453â†'m/z230for repaglinide, m/z130â†'m/z60for metformin, m/z458â†'m/z230for d5-repaglinide, and m/z136â†'m/z60for d6-metformin.Results The linear calibration curves for repaglinide and metformin were obtained in the concentration range of0.2-60.0ng·mL-1and4-1000ng·mL-1in plasma, respectively. The intraday and interday relative standard deviation (RSD) for repaglinide and metformin over the entire concentrations across validation runs were all less than11.5%, and relative error (RE) ranged from-1.3%to5.5%. The90%confidence interval of the test/reference geometric mean ratios of Cmax, AUC0-t, AUC0-∞for repaglinide were91.0%～116.2%,88.1%～98.8%,88.4%～99.2%, respectively. The90%confidence interval of the test/reference geometric mean ratios of Cma,> AUC0-t, AUC0-∞for metformin were96.4%～112.1%,92.8%～101.3%,92.6%～101.1%, respectively. The results demonstrated that the two formulations were bioequivalent.Conclusion This method has been used in the simultaneous determination of repaglinide and metformin in human plasma with stable labeled compounds as internal standards. It was suitable for the pharmacokinetic and bioequivalence study of repaglinide and metformin.