Synthesis and bioanalytical evaluation of morphine-3-O-sulfate and morphine-6-O-sulfate in human urine and plasma using LC-MS/MS.

The aim of this work was to synthesize morphine-3-O-sulfate and morphine-6-O-sulfate for use as reference substances, and to determine the sulfate conjugates as possible heroin and morphine metabolites in plasma and urine by a validated LC-MS/MS method. Morphine-6-O-sulfate and morphine-3-O-sulfate were prepared as dihydrates from morphine hydrochloride, in overall yields of 41 and 39% with product purities of >99.5% and >98%, respectively. For bioanalysis, the chromatographic system consisted of a reversed-phase column and gradient elution. The tandem mass spectrometer was operated in the positive electrospray mode using selected reaction monitoring, of transition m/z 366.15 to 286.40. The measuring range was 5-500 ng/mL for morphine-3-O-sulfate and 4.5-454 ng/mL for morphine-6-O-sulfate in plasma. In urine, the measuring range was 50-5000 ng/mL for morphine-3-O-sulfate and 45.4-4544 ng/mL for morphine-6-O-sulfate. The intra-assay and total imprecision (coefficient of variation) was below 11% for both analytes in urine and plasma. Quantifiable levels of morphine-3-O-sulfate in authentic urine and plasma samples were found. Only one authentic urine sample contained a detectable level of morphine-6-O-sulfate, while no detectable morphine-6-O-sulfate was found in plasma samples.