Simple, Sensitive, High-Throughput Method for the Quantification of Mitragynine in Rat Plasma Using UPLC-MS and Its Application to an Intravenous Pharmacokinetic Study

Abstract

A simple, sensitive and rapid ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) method was developed and validated for the quantification of mitragynine in rat plasma using amitriptyline hydrochloride as an internal standard. Sample preparation involved a one-step liquid–liquid extraction using methyl t-butyl ether. Mitragynine was separated on an Acquity UPLC™ BEH HILIC column using isocratic elution with a mobile phase of 10 mM ammonium formate buffer containing 0.1% formic acid:acetonitrile (15:85, v/v). At a flow rate of 0.2 mL min−1, the retention time of mitragynine was found to be 1.3 min. Ionization was performed in the positive ion electrospray mode. The selected mass-to-charge (m/z) ratio transition of mitragynine ion [M + H]+ used in the selected ion recording (SIR) was 399.1. The calibration curve was found to be linear over a concentration range of 1–5,000 ng mL−1 (r = 0.999) with a lower limit of quantification (LLOQ) of 1 ng mL−1. Intra- and inter-day assay variations were found to be less than 15%. The extraction recoveries ranged from 85–93% at the three concentrations (2, 400 and 4,000 ng mL−1) in rat plasma. This method was successfully used to quantify mitragynine in rat plasma following intravenous administration of the compound.