Quantification of morphine and metabolites in antemortem and postmortem human samples

Purpose: Morphine is one of the most effective agents for the short- and long-term control of significant pain. In humans, morphine is primarily metabolized to morphine-3-glucuronide (M3G) and, to a lesser extent to morphine-6-glucuronide (M6G). While M6G is a potent opioid receptor agonist with a higher analgesic activity, M3G has no opioid action and it seems to have a role in the side-effects usually described. Although several methods have been described for the simultaneous determination of morphine and its major metabolites, there is still need of methodologies with cleaner extraction, increased sensitivity, specificity and applicability to different matrices. Methods: A reversed-phase high-performance liquid chromatographic method with coulometric and diode-array detection was developed for the simultaneous determination of morphine, M3G and M6G in antemortem and postmortem samples, namely in plasma, serum, whole blood, urine, liver, kidney and brain. Morphine, glucuronides and the internal standard were extracted using Bond-Elut® C18 and Oasis® WCX solid-phase extraction cartridges and the separation was carried out by using a Waters Spherisorb® ODS2 reversed-phase column and 0.01 M potassium phosphate buffer:acetonitrile (85:15, v/v) containing sodium dodecyl sulfate as the mobile phase. Results: The method proved to be specific, accurate and precise across the calibration range with good linearity for all analytes. The quantification limit was set to 1 ng/ml for morphine and 10 ng/mL for M6G and M3G. The proposed method can be successfully applied in the quantification of morphine and metabolites, covering the routes of distribution, metabolism and elimination of morphine.