Determination of morphine, morphine-3-glucuronide and morphine-6-glucuronide in human serum by solid-phase extraction and liquid chromatography mass spectrometry with electrospray ionisation

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of morphine and two of its metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), in serum is described. The compounds are extracted from serum using Sep-Pak light C-18 solid-phase extraction cartridges, separated on an ODS C-18 analytical column (100 X 4.6 mm I.D.) and detected by electrospray ionisation mass spectrometry. The separation was achieved by running a linear gradient from 4 to 70% acetonitrile with formic acid added as modifier. The flow-rate in the column was 1.0 ml/min. After the column, the eluate was subjected to a 1:50 split. with 20 mu l/min delivered to the mass spectrometer and 980 mu l/min delivered to waste. The compounds were detected in the mass spectrometer by selected-ion monitoring for m/z 286.2 for morphine and 462.2 for M3G and M6G. The spray voltage was 2.4 kV and the sampling cone was set at 40 V. The compounds have been quantified in serum over a concentration range of 2.9-60 nmol/l (0.84-17 ng/ml) for morphine, 11-1080 nmol/l (5.0-500 ng/ml) for M3G and 4.3-220 nmol/l (2.0-100 ng/ml) for M6G using external standardisation. Intra-assay and inter-assay precision were in the range of 2.4-9.0% for all compounds. The major advantage with the present LC-MS method was the shorter analysis time, 10 min per sample compared to 45 min per sample with our previous LC method with dual detectors. The LC-MS method has proved to have both the selectivity and sensitivity needed for pharmacokinetic studies.