Pharmacokinetic and metabolism studies of rohitukine in rats by high performance liquid-chromatography with tandem mass spectrometry

Abstract

A sensitive, selective, and rapid high performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantification of rohitukine in rat plasma. HPLC was performed using a Symmetry-Shield C18 (5μ, 4.6×150mm) column, and isocratic elution with ammonium acetate buffer (pH4; 10mM):methanol (08:92, v/v) at a flow rate of 0.6mL/min. Sample clean-up involved solid phase extraction (SPE) of analyte and internal standard (phenacetin) from 100μL plasma. The parent→product ion transitions (MRM) for analyte and IS were 306.1→245.1m/z and 180.1→138.1m/z respectively, and were monitored on a triple quadrupole mass spectrometer, operating in positive ion mode. The method was validated across the dynamic concentration range of 5–500ng/mL for rohitukine, with a fast run time of 4.5min. The analytical method measured concentrations of rohitukine with accuracy (% bias) of <±10% and precision (% RSD) of <±12%. Rohitukine was stable during the battery of stability studies viz., bench-top, auto-sampler, freeze/thaw cycles and 30days of storage in a freezer at −70±10°C. Finally, the applicability of this assay has been successfully demonstrated in vivo pharmacokinetic and in vitro metabolism studies in Sprague–Dawley rat. This method will therefore be highly useful for future preclinical and clinical pharmacokinetic studies of rohitukine.