ORIGINAL ARTICLE Detection of morphine-3-sulfate and morphine-6-sulfate in human urine and plasma, and formation in liver cytosol Maria Andersson 1 , Linda Bj € orkhem-Bergman 2 , Lena Ekstr € om 1 , Lena Bergqvist 3 , Hugo Lagercrantz 3 , Anders Rane 1 & Olof Beck 1 1 Division of Clinical Pharmacology, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden 2 Division of Clinical Microbiology, Karolinska Institute, Stockholm, Sweden 3 Department of Woman and Child Health, Karolinska Institute, Stockholm, Sweden Keywords LC-MS/MS, morphine, morphine-3-sulfate, morphine-6-sulfate, plasma, urine Correspondence Maria Andersson, C2:78 Clinical Pharmacology, Karolinska University Hospital Huddinge and Karolinska Institute, SE-141 86 Stockholm, Sweden. Tel: +46-8-585-858-68; Fax: +46-8-585-810-70; E-mail: maria.ch. andersson@karolinska.se Funding Information This work was supported by grants from EU- FP7 NeoOpioid grant 223767; HEALTH-2007- 4.2-1. Received: 14 July 2014; Accepted: 17 July 2014 Pharma Res Per, 2(6), 2014, e00071, doi: 10.1002/prp2.71 doi: 10.1002/prp2.71 Abstract Morphine is still the mainstay in treatment of severe pain and is metabolized in the liver mainly by glucuronidation, partly to the pharmacologically active mor- phine-6-glucuronide (M6G). The sulfation pathway has attracted much less attention but may also form active metabolites. The aim of the present study was to study two sulfate metabolites of morphine in humans. Urine and plasma from newborns, adult heroin addicts, and terminal cancer patients was analyzed for the presence of morphine-3-sulfate (M3S) and morphine-6-sulfate (M6S) by a new liquid chromatography – tandem mass spectrometry (LC-MS/MS) method. In addition, morphine sulfation was studied in vitro in human liver cytosol preparations. M3S was present in urine and plasma from all study groups although at lower concentrations than morphine-3-glucuronide (M3G). The plasma M3S/M3G ratio was 30 times higher in newborns than in adults indicating that the relative sulfation is more important at early stage of life. M6S was measurable in only one plasma sample from a newborn patient, and in one of the urine sample from the drug testing group. The incubation of morphine with liver cytosol extracts resulted in approximately equal rate of for- mation of both M3S and M6S. In conclusion, sulfation of morphine is cata- lyzed in human liver but this minor metabolic pathway probably lacks clinical significance. The M6S metabolite is formed at a low rate, making it undetect- able in most individuals. Abbreviation LC-MS/MS, liquid chromatography – tandem mass spectrometry; LLOQ, lowest limit of quantification; M3G, morphine-3-glucuronide; M3S, morphine-3-sulfate; M6G, morphine-6-glucuronide; M6S, morphine-6-sulfate; M-d 3 , morphine-d 3 ; PAPS, 3 0 -phosphoadenosine 5 0 -phosphosulfate lithium salt hydrate; SULT, sulfo- transferase. Introduction Morphine has been used as a drug by humans for thou- sands of years for its analgetic effects. It is metabolized in the liver mainly by three different pathways, glucuronida- tion, sulfation, and N-demethylation (Milne et al. 1996; Aderjan and Skopp 1998; Projean et al. 2003). The main metabolic pathway is glucuronidation leading to the inac- tive morphine-3-glucuronide (M3G) and the pharmaco- logically active morphine-6-glucuronide (M6G). The human kinetics of morphine glucuronides have been thoroughly investigated ever since bioanalytical methods for their determination in blood became available (Svens- son et al. 1982; Milne et al. 1996). The sulfation pathway, on the other hand, has attracted much less attention. Although morphine-3-sulfate (M3S) is generally consid- ered to be present in humans, the presence of morphine- 6-sulfate (M6S) still remains to be demonstrated. The ª 2014 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. 2014 | Vol. 2 | Iss. 6 | e00071 Page 1