Development and validation of highly selective and robust method for simultaneous estimation of pioglitazone, hydroxypioglitazone and metformin in human plasma by LC–MS/MS: Application to a pharmacokinetic study

•Robust single method for distantly polar compounds in human plasma by LC–MS/MS.•Simple economic precipitation extraction procedure with low sample volume.•Labeled internal standards to track the analytes suitably.•Rapid analysis turnaround time for pharmacokinetic studies.•Negligible matrix interferences with good recovery. A simple, selective and robust reverse phase high performance liquid chromatography–electrospray ionization-tandem mass spectrometry (ESI-MS/MS) method for simultaneous quantitation of pioglitazone (PIO), pioglitazone metabolite M-IV – hydroxypioglitazone (OH-PIO) and metformin (MET) in human plasma using deuterated internal standards (IS) is developed and fully validated as per industrial practices. After acetonitrile-induced protein precipitation of the plasma samples; PIO, OH-PIO, MET and IS were chromatographed on reverse phase column and analyzed in the multiple reaction monitoring in positive ion mode. The ion transitions were monitored at m/z 357.2→134.2 for PIO, 373.0→150.1 for OH-PIO, 130.2→71.0 for MET, 361.1→134.2 for PIO-IS and 136.1→77.1 for MET-IS. The total chromatographic run time was 4.0min. A linear response function (r>0.998) was established for the range of concentrations 15–2500ng/mL, 10–1500ng/mL and 25–3000ng/mL for PIO, OH-PIO and MET respectively in human plasma. The intra and inter-day precision and accuracy values have met the set acceptance criteria. The method is simple, selective, robust economic and has been applied successfully to more than 2000 plasma samples as part of pharmacokinetic study in humans.