Simultaneous Determination of Heroin, 6-Monoacetylmorphine, Morphine, and its Glucuronides by Liquid Chromatography-Atmospheric Pressure Ionspray-Mass Spectrometry

A new analytical technique has been developed for the simultaneous determination of heroin, 6-monoacetylmorphine, morphine, morphine-6- and 3-glucuronides, and codeine in serum using liquid chromatography coupled with ionspray mass spectrometry. The analytes and the internal standard, nalorphine, were subjected to solid-phase extraction (SPE) using ethyl SPE columns before chromatography. The chromatographic separation of the analytes was achieved using a normal phase column and a water-methanol-acetonitrile-formic acid mobile phase at a flow rate of 230 µL/min. The mass spectrometer was operated in selected-ion monitoring mode. Under these conditions, the limit of quantitation was 0.5 ng/mL for heroin, 4 ng/mL for 6-monoacetylmorphine, 4 ng/mL for morphine, 1 ng/mL for morphine-3-glucuronide, 4 ng/mL for morphine-6-glucuronide, and 4 ng/mL for codeine. Serum levels of heroin metabolites were determined in C57BL/6 inbred mice after a dose of 20 mg/kg heroin administered subcutaneously. 6-Monoacetylmorphine showed a peak concentration of 0.93 µg/mL serum at 3 min, whereas morphine and morphine-3-glucuronide achieved their peak concentrations of 9.6 and 2.9 µg/mL serum at 10 and 20 min, respectively. Finally, the absence of morphine-6-glucuronide and codeine excluded the possibility of their formation from morphine in this animal model.