Journal of Chromatography B, 876 (2008) 137–142 Contents lists available at ScienceDirect Journal of Chromatography B journal homepage: www.elsevier.com/locate/chromb Short communication Detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography–tandem mass spectrometry Mark C. Parkin a , Sophie C. Turfus a , Norman W. Smith a , John M. Halket a , Robin A. Braithwaite b , Simon P. Elliott b , M. David Osselton c , David A. Cowan a , Andrew T. Kicman a,∗ a Department of Forensic Science & Drug Monitoring, King’s College London, 150 Stamford Street, London SE1 9NH, UK b The Regional Laboratory for Toxicology, City Hospital, Dudley Road, Birmingham B18 7QH, UK c The Forensic Science Service, Trident Court 2920, Birmingham Business Park, Birmingham B37 7YN, UK article info Article history: Received 3 June 2008 Accepted 29 September 2008 Available online 4 October 2008 Keywords: Ketamine Metabolites Drug facilitated sexual assault Liquid chromatography Mass spectrometry abstract Current analytical methods used for screening drugs and their metabolites in biological samples from victims of drug-facilitated sexual assault (DFSA) or other vulnerable groups can lack sufficient sensitivity. The application of liquid chromatography, employing small particle sizes, with tandem mass spectrometry (MS/MS) is likely to offer the sensitivity required for detecting candidate drugs and/or their metabolites in urine, as demonstrated here for ketamine. Ultra-performance liquid chromatography–mass spectrometry (UPLC–MS/MS) was performed following extraction of urine (4 mL) using mixed-mode (cation and C8) solid-phase cartridges. Only 20 L of the 250 L extract was injected, leaving sufficient volume for other assays important in DFSA cases. Three ion transitions were chosen for confirmatory purposes. As ketamine and norketamine (including their stable isotopes) are available as reference standards, the assay was additionally validated for quantification purposes to study elimination of the drug and primary metabo- lite following a small oral dose of ketamine (50mg) in 6 volunteers. Dehydronorketamine, a secondary metabolite, was also analyzed qualitatively to determine whether monitoring could improve retrospec- tive detection of administration. The detection limit for ketamine and norketamine was 0.03ng/mL and 0.05 ng/mL, respectively, and these compounds could be confirmed in urine for up to 5 and 6 days, respec- tively. Dehydronorketamine was confirmed up to 10 days, providing a very broad window of detection. © 2008 Published by Elsevier B.V. 1. Introduction Ketamine [2-(2-chlorophenyl)-2-methylaminocyclohexan-1- one] (Fig. 1) is a N-methyl-d-aspartate receptor antagonist used legitimately for its dissociative anaesthetic properties [1,2]. Recre- ational use of ketamine has gained popularity worldwide over the last 10 years [3,4], with users experiencing hallucinations and a cataleptic state called the ‘K-hole’ [5,6]. The cataleptic state brought on by administration of ketamine can also produce stupor and sedation, that together with possible amnesia and difficulty in fighting off an assailant, has led to its recent implication in drug-facilitated sexual assault (DFSA) [7,8]. DFSA incidents are often reported later than 24 h after the alleged assault, when very little drug will remain in the victim. Similarly, late samples are often collected from children where suspicion of non-accidental poisoning may only come to light after routine clin- ∗ Corresponding author. Tel.: +44 20 7848 4779; fax: +44 20 7848 4980. E-mail address: andrew.kicman@kcl.ac.uk (A.T. Kicman). ical evaluation has been performed. This has led to several groups reporting the use [9,10] and need [11,12] for highly sensitive assays that can provide sufficient retrospective detection for ketamine together with other classes of fast acting sedative drugs. Many procedures have been described for detecting ketamine and its active metabolite norketamine in human urine, with vary- ing limits of detection, ranging from 0.5 ng/mL to 25 ng/mL for both compounds [13–19] but only one group describe a method with an LOD of 0.05 ng/mL for norketamine, which was used to assay samples following intravenous infusion of ketamine for therapeu- tic purposes [13]. Despite these investigations on the analysis of ketamine, there is a paucity of data regarding the urinary elimina- tion of ketamine and norketamine following oral administration. We developed a highly sensitive quantitative assay for ketamine and its primary metabolite, norketamine, in urine and evaluated its applicability for forensic purposes by performing a low-dose elimi- nation study. Dehydronorketamine was not available as a standard at the time of investigation but as Cheng et al. [20] indicated that it may be an important diagnostic marker, we chose to perform qualitative analysis for this metabolite. 1570-0232/$ – see front matter © 2008 Published by Elsevier B.V. doi:10.1016/j.jchromb.2008.09.036