An improved extraction method for the HPLC determination of morphine and its metabolites in plasma

A new, simple and rapid extraction procedure coupled with a combined coulometric-fluorescence HPLC assay is described for the simultaneous determination of morphine (M) and morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), and normorphine (NM) in plasma. The effect of concentration and pH of selected ion-pairing agents on the extraction of these compounds from plasma by solid-phase extraction was investigated. The extraction procedure was optimized in terms of recovery, reproducibility and lack of interference from endogenous materials. The optimized method uses tetrabutylammonium hydrogen sulphate (TBAHS) at pH 10 followed by separation on a single C18 solid-phase extraction cartridge. For routine analysis the procedure provides high and reproducible recoveries over a concentration range of 1.0-1000 ng ml-1 for morphine, M6G and normorphine and 20-1000 ng ml-1 for M3G. The method was used successfully to analyse plasma samples from a pharmacokinetic study in which sheep had received an intravenous dose of 0.015 mg kg-1 of M6G.