A Stereospecific Microassay for the Determination of Morphine-6-β-D-Glucuronide and other Active Morphine Metabolites in the Neonatal Guinea Pig

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Abstract
A stereospecific microassay for the simultaneous determination of morphine and active metabolites morphine-6-β-D-glucuronide, morphine-3-β-D-glucuronide and normorphine using reversed-phase high-performance liquid-chromatography with ultraviolet detection is described. Solid-phase extraction was followed by chromatography on an octadecylsilica column eluted by 16.5% acetonitrile in phosphate buffer, pH 2.1 with sodium dodecyl sulfate as an ion-pairing agent. Using an 80 μl sample size the limits of quantitation were 94 ng/ml for all drugs. Coefficients of variation ranged from 2.0% to 6.3%. The assay is specific and separates the stereoisomers morphine-6-α-D-glucuronide and morphine-6-β-D-glucuronide. This microassay has been applied to samples of guinea pig plasma, following subcutaneous injections of morphine, and to hepatic microsomal enzyme preparations from a pregnant guinea pig. Neonatal guinea pigs produce substantial amounts of morphine-3-glucuronide and morphine-6-glucuronide from morphine in vivo, but normorphine was not detectable, whereas the liver microsomal enzyme preparations from a pregnant dam rapidly produced normorphine.