Quantitation of Morphine, Morphine-3-Glucuronide, and Morphine-6-Glucuronide in Plasma and Cerebrospinal-Fluid Using Solid-Phase Extraction and High-Performance Liquid-Chromatography with Electrochemical Detection

An original, sensitive, and specific high-performance liquid chromatographic (HPLC) assay was developed for the quantitation of morphine and its two major metabolites, morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G), in human plasma and cerebrospinal fluid (CSF) and in rat plasma, using hydromorphone as the internal standard. Solid-phase extraction was used to separate morphine and its glucuronide metabolites from plasma constituents. Extraction efficiencies of morphine, M3G, and M6G from human plasma samples (0.5 ml) were 84, 87, and 88%, respectively. Extraction efficiencies of morphine, M3G, and M6G did not differ significantly (p > 0.05) between human plasma and CSF or rat plasma. Morphine, M3G, M6G, and hydromorphone were separated on a 10 mu C-8 Resolve radially compressed cartridge using a mobile phase comprising methanol:acetonitrile:phosphate buffer, (0.0125M pH 7.5; 10: 10:80), in which 11 mg/L of cetyltrimethylammonium bromide (cetrimide) was dissolved. Quantitation was achieved using a single electrochemical detector at ambient temperature (23-degrees-C). Standard curves were linear over the ranges 0.020-2.190, 0.027-2.709, and 0.027-0.542 muM for morphine, M3G, and M6G, respectively. Lower limits of detection for morphine, M3G, and M6G in human plasma and CSF samples (0.5 ml) were 0.020, 0.027, and 0.027 muM, respectively. Corresponding lower limits of detection in rat plasma (0.1 ml) were 0.102, 0.135, and 0.135 muM, respectively. Intraassay precision for low and high concentrations of morphine, M3G, and M6G wer