Simultaneous quantification of capsaicin and dihydrocapsaicin in rat plasma using HPLC coupled with tandem mass spectrometry

A rapid, simple and sensitive HPLC–ESI–MS/MS method was developed for the simultaneous determination of capsaicin and dihydrocapsaicin in rat plasma. Plasma samples containing capsaicin, dihydrocapsaicin and phenacetin (internal standard) were prepared based on a simple protein precipitation by the addition of two volumes of acetonitrile. The analytes and internal standard were separated on a Zorbax SB-C18 column (3.5 μm, 2.1 mm × 100 mm) with mobile phase of acetonitrile/water (55:45, v/v) containing 0.1% formic acid (v/v) at a flow rate of 0.2 mL/min with an operating temperature of 25 °C. Quantification was performed on a triple quadrupole mass spectrometer equipped with electrospray ionization (ESI) source by selected reaction monitoring (SRM) of the transitions at m/z 306–137 for capsaicin, m/z 308–137 for dihydrocapsaicin and m/z 180–110 for the IS. Linear detection responses were obtained for capsaicin and dihydrocapsaicin ranging from 1 to 500 ng/mL and the lower limits of quantitation (LLOQs) for the two compounds were 1 ng/mL. The intra- and inter-day precisions (R.S.D.%) were within 9.79% for the two analytes, while the deviations of assay accuracies were within ±10.63%. The average recoveries of the analytes were greater than 89.88%. The analytes were proved to be stable during all sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic studies of capsaicin and dihydrocapsaicin in rats after subcutaneous administration of capsaicin (natural, containing 65% capsaicin and 35% dihydrocapsaicin).