A Highly Sensitive Assay for the Simultaneous Determination of Morphine, Morphine-3-Glucuronide, and Morphine-6-Glucuronide in Human Plasma by High-Performance Liquid Chromatography with Electrochemical and

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Abstract
Summary A novel, highly sensitive and specific bioanalytical method has been developed for the simultaneous determination of morphine and its major metabolites, morphine-3-glucuronide and morphine-6-glucuronide, in human plasma, using noroxymorphone as the internal standard. The analytes are isolated from human plasma using a nonpolar/polar C2 solid-phase extraction cartridge and analyzed by high-performance liquid chromatography with serial detection using electrochemical detection for morphine, morphine-6-glucuronide (M6G), and noroxymorphone and fluorescence detection for morphine-3-glucuronide (M3G). The limit of quantitation (sensitivity) using a 0.5-ml sample of plasma is 1 ng/ml, 10 ng/ml, and 5 ng/ml for morphine, M3G, and M6G, respectively. Standard curves were linear (correlation coefficients >0.999) over the ranges 1-30 ng/ml, 10-500 ng/ml, and 5-100 ng/ml for morphine, M3G, and M6G, respectively. The overall interday accuracy of the method was -1.58% for morphine, 2.27% for M3G, and -5.34% for M6G. The assay is routinely used for the study of morphine M3G, and M6G pharmacokinetics after oral administration of morphine.