Aims: To characterize directly the conjugated metabolites of morphine in urine samples of cancer patients.
Methods: Urine samples from the patients were treated by solid-phase extraction method and chromatographed using three high-performance liquid chromatography systems. Conjugated metabolites were directly detected with liquid chromatographic/ion trap mass spectrometric (LC/MSn) technique by selected ion monitoring, full scan MS/MS and MS3 modes.
Results: Six conjugated metabolites including two new metabolites M5 and M6 were found. Morphine-3-glucuronide (M-3-G) and morphine-6-glucuronide (M-6-G) were identified by comparing their l.c. retention times and multistage mass spectra with those of the reference substances. Two novel metabolites, morphine-3-glucoside and morphine-6-glucoside, as well as normorphine glucuronides were identified by comparing their mass fragment patterns and l.c. retention times with those of M-3-G and M-6-G. Hydrolysis of urine samples with beta-glucosidase and beta-glucuronidase provided further evidence of the metabolites M5 and M6 as morphine glucosides. The excretion amounts of morphine conjugates in urines were in the order of morphine glucuronides, morphine glucosides and normorphine glucuronides.
Conclusions: In the present study, the applications of l.c. separation and multistage mass spectra have permitted the direct identification of conjugated metabolites of morphine. To our knowledge, this is the first report about O-linked glucosides of morphine at 3-aromatic and 6-aliphatic hydroxyl groups.