Ion-pair solid phase extraction for the simultaneous separation and quantitation of metformin and canagliflozin in human plasma by LC-MS/MS

A high-throughput and sensitive liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) has been developed for the simultaneous determination of metformin and canagliflozin in human plasma. The optimized ion-pair solid phase extraction of the analytes was carried out using 100 mu L plasma, followed by reversed-phase chromatography on COSMOSIL 5CN-MS (150 x 4.6 mm, 5 mu m) column. An isocratic mobile phase consisting of methanol-20 mM ammonium formate in water, pH 5 (75:25, v/v) was used for separation of the analytes with a resolution factor of 2.18. Mass spectrometer was operated in the multiple reaction monitoring mode at unit mass resolution. Electro spray ionization was used for mass transition of m/z 130.1/60.1 and m/z 462.1/267.1 for metformin and canagliflozin, respectively. The method was successfully validated for the concentration range of 2.50-2500 ng/mL for both the analytes. The intra- and inter-batch precision determinations for metformin and canagliflozin, expressed as coefficient of variation were lower than 5.5% at all the test concentrations. Recovery of both the drugs was in the range of 78-90% and was reproducible in the quality control samples. No significant matrix effects were observed for the analytes as evident from the IS normalized matrix factors. Stability of the drugs in whole blood and plasma samples was ascertained under different conditions. The assay showed acceptable performance when applied to clinical samples generated from healthy Indian subjects.