Determination of tolperisone in human plasma by liquid chromatography/tandem mass spectrometry for clinical application

We have developed and validated a simple, rapid, and sensitive liquid chromatography analytical method employing tandem mass spectrometry (LC–MS/MS) for the determination of tolperisone, a centrally acting muscle relaxant, in human plasma. After liquid–liquid extraction with methyl t-butyl ether, chromatographic separation of tolperisone was performed using a reversed-phase Luna C18 column (2.0 mm × 50 mm, 5 μm particles) with a mobile phase of 10 mM ammonium formate buffer (pH 3.5) – methanol (12:88, v/v) and quantified by tandem mass detection in ESI positive ion mode. The flow rate of the mobile phase was 250 μL/min and the retention times of tolperisone and the internal standard (IS, dibucaine) were both 0.6 min. The calibration curves were linear over a range of 0.5–300 ng/mL (r > 0.999). The lower limit of quantification, using 200 μL human plasma, was 0.5 ng/mL. The mean accuracy and precision for intra- and inter-day validation of tolperisone were within acceptable limits. The LC–MS/MS method reported here showed improved sensitivity for quantification of tolperisone in human plasma compared with previously described analytical methods. Lastly, the validated method was successfully applied to a pharmacokinetic study in humans.