Simultaneous determination of thienorphine and its active metabolite thienorphine-glucuronide in rat plasma by liquid chromatography–tandem mass spectrometry and its application to pharmacokinetic studies

Abstract: A simple, sensitive and reliable method was developed to determine simultaneously the concentrations of thienorphine and its metabolite thienorphine glucuronide conjugate in rat plasma by liquid chromatography–tandem mass spectrometry (LC–MS/MS). The metabolite was identified by MS: thienorphine glucuronide conjugate. Sample preparation involved protein precipitation with methanol. Analytes were separated on Finnigan BetaBasic-18 column (150mm×2.1mm i.d., 5μm) using methanol: water: formic acid (56:44:0.1, v/v/v) as mobile phase at a flow rate of 0.2ml/min. The method had a linear calibration curve over the concentration range of 0.1–50ng/ml for thienorphine and 2–1000ng/ml for thienorphine glucuronide conjugate, respectively. LOQ of thienorphine and thienorphine glucuronide conjugate was 0.1 and 2ng/ml, respectively. The intra- and inter-batch precisions were less than 12% and their recoveries were greater than 80%. Pharmacokinetic data of thienorphine and its metabolite thienorphine glucuronide conjugate obtained with this method following a single oral dose of 3mg/kg thienorphine to rats were also reported for the first time. [Copyright &y& Elsevier]