Bioanalytical method development and validation using incurred samples: Quantitative estimation of metformin in human K3EDTA plasma by LC–MS/MS

Abstract

Bioanalytical methods should be reproducible and consistent when applying to clinical sample analysis. Incurred sample reanalyses (confirmatory reanalyses) are performed to support clinical data, and regulatory agencies evaluate the same before approval of bioequivalent products/drugs. A confirmatory reanalysis was demonstrated for metformin after administration of 2/500 mg repaglinide + metformin fixed dose formulation under fasted and fed conditions. The liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of metformin in human plasma using metformin-d₆as an internal standard has been developed and validated. The ions transitions recorded in multiple reaction monitoring (MRM) were m/z 130.1→60.0 and 136.2→60.0 for metformin and metformin-d₆, respectively. The compounds were isolated by solid phase extraction and separated on a C12 reverse phase (Synergi MAX-RP 80A) column, using isocratic mobile phase flow at a flow rate of 0.8 mL/min. No matrix effect was observed within the linearity range of 10.2–1741.8 ng/mL (r² > 0.99). The acceptable result of confirmatory reanalysis further indicated stability of metformin in the presence of repaglinide. The assay method was found to be highly reproducible and was successfully applied for pharmacokinetic evaluations of metformin in fixed dose combinations with repaglinide.

Keywords::

• LC–APCI-MS/MS
• metformin method validation
• incurred sample reanalysis
• clinical sample analysis reproducibility

Acknowledgments

The authors wish to acknowledge the support and facilities received from Ranbaxy Research Laboratories, Gurgaon, India, for carrying out this work.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.