Determination of Morphine, Fentanyl and Their Metabolites in Small Sample Volumes Using Liquid Chromatography Tandem Mass Spectrometry.

The USA and numerous other countries worldwide are currently experiencing a public health crisis due to the abuse of heroin and illicitly manufactured fentanyl. We have developed a liquid chromatography tandem mass spectrometry (LC-MS-MS)-based method for the detection of morphine, fentanyl and their metabolites, including morphine-3-glucuronide (M3G), morphine-6-glucuronide (M6G), normorphine, norfentanyl and deuterated internal standards in limited sample volumes with the limit of detection of 5.0/0.5 ng/mL (morphine, M3G, M6G, normorphine/fentanyl, norfentanyl). The inter-assay precision (%CV) was less than 12% for all assays, and the inter-assay bias (%) was less than 5%. The ruggedness of the method, dilution effect and carryover were also investigated as part of the study. The simultaneous quantification of morphine, fentanyl and its metabolites via this simple and time- and cost-efficient method could be successfully applied to samples taken for pharmacokinetic evaluation (antemortem and postmortem) after a single dose of morphine or co-administration of morphine with other drugs (e.g., fentanyl) in rats.

A Mechanism-Based Forensic Investigation into the Postmortem Redistribution of Morphine.

The interpretation of postmortem drug levels is complicated by the change in drug blood levels during the postmortem period, a phenomenon known as postmortem drug redistribution. We investigated the postmortem redistribution (PMR) of morphine, morphine-3-glucuronide and normorphine in the rat. Morphine (10 mg/kg) was intravenously injected into rats, followed by euthanasia 1 h post-injection. The carcasses were placed in a supine position at room temperature, and tissues including heart blood, femoral blood, liver, lung and brain were collected at different time points: 0, 8, 16 or 24 h postmortem. The samples were analyzed with a validated (following modified Scientific Working Group for Forensic Toxicology (SWGTOX) (20) guidelines) liquid chromatography-tandem mass spectrometry method. The use of a mechanism-based approach (involving the used set doses of drug with the study performed in controlled environment) to assess PMR using systematic and statistical analyses provides important information that has not previously been presented in PMR literature. While previous human studies focus on central to peripheral ratios as well as peripheral to tissue ratio, this work focused on the change in morphine and metabolite concentrations over the course of the postmortem interval in relation to each other in addition to the comparison to additional matrices at each postmortem interval. Postmortem redistribution was identified in several tissues across the postmortem interval; however, there was minimal statistical difference observed among each matrix at a given postmortem interval with the exception of normorphine and morphine-3-glucuronide. Combined, our study provides a valuable resource and reference information that can aide toxicologists, medical examiners or coroners when assessing postmortem drug concentrations of morphine and metabolites when they are making determinations of cause of death.

Formation of benzoylecgonine isopropyl ester following solid-phase extraction.

In forensic toxicology general alkaline drug screens typically utilize liquid liquid [LLE] or solid phase extraction [SPE] sample preparation techniques. It is expected that different drugs will be detected when a laboratory changes techniques. In this study, when the authors switched from LLE to SPE they were able to detect benzoylecgonine [BE]. Benzoylecgonine isopropyl ester [BEIE] was also detected. Further investigation demonstrated that the BEIE was formed during sample elution with methylene chloride/isopropanol/ammonium hydroxide. BEIE was not detected if methanol/ammonium hydroxide was used as the elution solvent. Analysts should be aware that BEIE is formed in the presence of BE if elution solvents comprise isopropanol and a strong base. Therefore, use of BEIE as an internal standard in such assays will result in inaccurate quantitation of BE.