Simultaneous determination of flupirtine and its major active metabolite in human plasma by liquid chromatography-tandem mass spectrometry

A rapid, selective and sensitive HPLC-tandem mass spectrometry method was d eveloped and validated for simultaneous determination of flupirtine and its active metabolite D-13223 in human plasma. The analytes and internal stand ard diphenhydramine were extracted from plasma samples by liquid-liquid ext raction, and chromatographed on a C-18 column. The mobile phase consisted o f acetonitrile-water-formic acid (60:40:1, v/v/v), at a flow rate of 0.5 ml /min. Detection was performed on a triple quadrupole tandem mass spectromet er by selected reaction monitoring (SRM) mode via atmospheric pressure chem ical ionization (APCI). The method has a limit of quantitation of 10 ng/ml for flupirtine and 2 ng/ml for D-13223, using 0.5-ml plasma sample. The lin ear calibration curves were obtained in the concentration range of 10.0-150 0.0 ng/ml for flupirtine and 2.0-300.0 ng/ml for D-13223. The intra- and in ter-run precision (RSD), calculated from quality control (QC) samples was l ess than 7.2% for flupirtine and D-13223. The accuracy as determined from Q C samples was less than 5% for the analytes. The overall extraction recover ies of flupirtine and D-13223 were determined to be about 66% and 78% on av erage, respectively. The method was applied for the evaluation of the pharm acokinetics of flupirtine and active metabolite D-13223 in volunteers follo wing peroral administration. (C) 2001 Elsevier Science B.V. All rights rese rved.