Simultaneous determination of morphine and its glucuronides in rat hair and rat plasma by reversed-phase liquid chromatography with electrospray ionization mass spectrometry

Abstract

The simultaneous determination of morphine and the glucuronide metabolites [morphine-3-β-D-glucuronide (M3G) and morphine-6-β-D-glucuronide (M6G)] in rat hair and rat plasma was carried out using reversed-phase high-performance liquid chromatography (HPLC) coupled with electrospray ionization mass spectrometry (ESI-MS). The chromatographic separation of the analytes was achieved using a semi-micro-HPLC column (3 μm particle size; 100 × 2.0 mm id) by gradient elution with 50 mM ammonium acetate and acetonitrile as eluents. After separation, morphine and the glucuronides were determined by selected ion monitoring (SIM) of ESI-MS using the quasi-molecular ions [M + H]⁺ at m/z = 286 and 462, respectively. The calibration curves were linear between the concentration of the analytes and the deuterium-labelled morphine (M-d₃) selected as internal standard. The method was applied for the determination of the incorporation of morphine and the glucuronides into the hair shafts and hair roots of Dark Agouti rats after single intraperitoneal administration of morphine hydrochloride. Plasma concentrations of morphine and glucuronides were simultaneously determined after administration. Morphine and M3G were detected in the hair shafts and the hair roots. The concentrations of M3G in the hair root were lower than those of morphine in all sampling periods. In contrast, M3G concentrations in plasma were relatively higher at each sampling time. Small quantities of M6G were also identified in the plasma up to 4 h after administration. The concentration difference between the hair root and plasma seems to be due to the incorporation ratio of morphine and glucuronide into hair. As M3G was also identified in the hair shaft 1 week after administration, the incorporation of glucuronide metabolites into hair is obvious. This is the first report of the identification of morphine glucuronide in hair samples without the use of acid hydrolysis or enzyme digestion.