Validation of an LC–MS/MS Method for Simultaneous Detection of Five Selective KCNQ Channel Openers: ICA-27243, ML-213, PF-05020182, SF-0034 and Flupirtine in Mice Plasma and its Application to a Pharmacokinetic Study in Mice

A sensitive and rapid LC–MS/MS method was developed and validated for the simultaneous quantitation of five selective KCNQ channel openers, namely ICA-27243, ML-213, PF-05020182, SF-0034 and flupirtine in mice plasma as per regulatory guideline. The analytes and the internal standard (IS; flupirtine-d 4 ) were extracted from 50 µL mice plasma by liquid–liquid extraction, followed by chromatographic separation using an Atlantis C18 column with an isocratic mobile phase comprising 0.2% formic acid: acetonitrile (20:80, v/v) at a flow rate of 0.6 mL min−1 within 2.5 min. Detection and quantitation was done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 268.9 → 140.8, 258.1 → 95.1, 367.2 → 269.1, 322.2 → 248.2, 305.7 → 196.4 and 309.1 → 196.1 for ICA-27243, ML-213, PF-05020182, SF-0034, flupirtine and the IS, respectively, in the positive ionization mode. The calibration curves were linear from 1.00 to 2008 ng mL−1 for all the analytes with r2 ≥ 0.99. The intra- and inter-batch accuracy and precision (% CV) across quality controls varied from 90.0 to 113 and 2.64 to 13.0; 93.8 to 114 and 3.15 to 14.9%, respectively, for all the analytes. Analytes were found to be stable under different stability conditions. The method was applied to a pharmacokinetic study in mice.