Quantification of morphine and its major metabolites M3G and M6G in antemortem and postmortem samples

Morphine is one of the most effective agents for the control of significant pain, primarily metabolized to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). While M6G is a potent opioid agonist, M3G has no opioid action and seems to have a role in side-effects caused by morphine. In this study, a reversed-phase high-performance liquid chromatographic method with diode-array and electrochemical detection was developed for the simultaneous determination of morphine, M3G and M6G in antemortem and postmortem samples (plasma, whole blood, urine, liver, kidney and brain). Morphine, glucuronides and internal standard were extracted by double solid-phase extraction and the separation was carried out with a Waters Spherisorb® ODS2 reversed-phase column and potassium phosphate buffer (pH = 2.2)–acetonitrile containing sodium dodecyl sulfate as the mobile phase. The method proved to be specific with good linearity for all analytes in a calibration range from 1 to 600 ng/mL and proved to be accurate and have adequate precision and recovery. Limits of detection in the studied matrices were 0.4–4.5 ng/mL for morphine, 2.7–6.1 ng/mL for M3G and 0.8–4.4 ng/mL for M6G. The proposed method can be successfully applied to quantify morphine and its metabolites in several biological samples, covering the major routes of distribution, metabolism and elimination of morphine.