High-performance liquid chromatography-mass spectrometry-mass spectrometry analysis of morphine and morphine metabolites and its application to a pharmacokinetic study in male Sprague-Dawley rats

A high-performance liquid chromatography tandem mass spectrometry-mass spectrometry (LC-MS-MS) assay was developed for the analyses of morphine, morphine glucuronides and normorphine in plasma samples from rats. The analytes were extracted by using C2 solid-phase extraction cartridges. The extraction recoveries were 100% for morphine, 84% for morphine-3-glucuronide, 64% for morphine-6-glucuronide and 88% for normorphine. Both intra- and inter-assay variabilities were below 11%. Using a plasma sample size of 100 microliters, the limits of detection were 13 nmol l-1 (3.8 ng ml-1) for morphine, 12 nmol l-1 (5.5 ng ml-1) for morphine-3-glucuronide, 26 nmol l-1 (12 ng ml-1) for morphine-6-glucuronide and 18 nmol l-1 (5.0 ng ml-1) for normorphine, at a signal-to-noise ratio of 3. The present assay was applied to a pharmacokinetic study in rats after intraperitoneal administration of morphine.