Quantification of morphine and its major metabolites M3G and M6G in antemortem and postmortem samples Ana Oliveira a,b,c *, Félix Carvalho a , Paula Guedes Pinho a , Fernando Remião a , Rui Medeiros b,c,d,e,f and Ricardo Jorge Dinis-Oliveira a,c,g ABSTRACT: 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 car- ried 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. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: morphine; morphine-3-glucuronide; morphine-6-glucuronide; metabolism; HPLC-DAD-electrochemical Introduction Morphine, an alkaloid present in the poppy plant, is the main- stay of pharmacological treatment for moderate-to-severe acute and chronic cancer-related pain (WHO, 1996; Ross et al., 2005). However, despite its widespread clinical use, this opioid displays wide variations in its pharmacological efficacy and tolerability, presenting some side-effects that can compromise the patient safety/compliance and its analgesic effectiveness (Dinis-Oliveira et al., 2012a, 2012b). Morphine is extensively metabolized in the human liver especially by UDP-glucuronosyltransferase 2B7 (UGT2B7) producing two important metabolites, morphine-6-glucuronide (M6G; 10–15%) and morphine-3-glucuronide (M3G; 45–55%), by glucuronidation of the 6-OH alcoholic group and the 3-OH phenolic group, respec- tively (Fig. 1; Carrupt et al., 1991). Other UGT isoforms seem to be involved in M3G formation, like UGT1A3, 1A6, 1A8, 1A9 and 1A10 (Stone et al., 2003). M6G is a potent opioid receptor agonist with higher analgesic activity as compared with morphine (Carrupt et al., 1991; Osborne et al., 1992). M3G has no opioid action and it seems to have a role in the side-effects usually described, namely hyperalgesia/allodynia, neurotoxicity and an antagonistic effect, decreasing morphine analgesia (Carrupt et al., 1991; Christrup, 1997; Holthe et al., 2002). A variability of metabolites formation has been described in humans (Holthe et al., 2002; Sawyer et al., 2003; Klepstad et al., 2005) and the different roles played by each compound may also account for different pain intensities and morphine requirements (Klepstad et al., 2000). Therefore, the quantification of morphine simulta- neously with its glucuronide metabolites and the calculation of metabolic ratios have become of increasingly interest for a better understanding of morphine efficacy and side-effects, as well as for the interpretation of toxic deaths involving heroin or morphine (Staub et al., 1990; Bosch et al., 2007). * Correspondence to: A. Oliveira, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal. Email: anacorreiadeoliveira@gmail.com a REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal b Molecular Oncology GRP and Virology LB, Portuguese Institute of Oncology- Porto, Porto, Portugal c IINFACTS – Institute of Research and Advanced Training in Health Sciences and Technologies, Department of Sciences, Advanced Institute of Health Sciences – North (ISCS-N), CESPU, CRL, Gandra, Portugal d ICBAS, Abel Salazar Institute for the Biomedical Sciences, Porto, Portugal e Faculty of Health Sciences of Fernando Pessoa University, Porto, Portugal f LPCC, Portuguese League Against Cancer, Regional Centre of the North, Porto, Portugal g Department of Legal Medicine and Forensic Sciences, Faculty of Medicine, University of Porto, Porto, Portugal Abbreviations used: M3G, morphine-3-glucuronide; M6G, morphine-6- glucuronide; SPE, solid-phase extraction; UGT2B7, UDP-Glucuronosyltransferase 2B7 Biomed. Chromatogr. 2014; 28: 1263–1270 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 26 October 2013, Revised: 9 January 2014, Accepted: 16 January 2014 Published online in Wiley Online Library: 20 February 2014 (wileyonlinelibrary.com) DOI 10.1002/bmc.3158 1263