Characterization of xylazine metabolism in rat liver microsomes using liquid chromatography–hybrid triple quadrupole– linear ion trap–mass spectrometry David St-Germain Lavoie a , Floriane Pailleux a,b , Pascal Vachon c and Francis Beaudry a * ABSTRACT: Xylazine is an a 2 -adrenoceptor agonist and it is widely used in veterinary anesthesia in combination with ketamine. There is limited information on the metabolism of xylazine. A quantitative method for the determination of xylazine by HPLC-ESI/MS/MS was developed. The method consisted of a protein precipitation extraction followed by analysis using liquid chromatography electrospray tandem mass spectrometry. The chromatographic separation was achieved using a Thermo Betasil Phenyl 100  2 mm column combined with an isocratic mobile phase composed of acetonitrile, methanol, water and formic acid (60:20:20:0.4) at a flow rate of 300 mL/min. The mass spectrometer was operating in selected reaction monitoring mode and the analytical range was set at 0.05–50 mM. The precision (%CV) and accuracy (%NOM) observed were 2.3–7.2 and 88.2–96.4%. In vitro metabolism studies were performed in rat liver microsomes and results showed moderate cytochrome P450 affinity (K m = 10.1 mM) and a low metabolic stability of xylazine with a half-life of 4.1 min in rat liver microsomes. Five phase 1 metabolites were observed. The main metabolite observed was an oxidation of the thiazine moiety at m/z 235 and, to a lesser extent, we observed the formation of N-(2,6-dimethylphenyl)thiourea at m/z 181 and three distinctive hydroxylated metabolites at m/z 237. Further experiments with ketamine and ketoconazole strongly supported that the metabolism of xylazine to its main metabolite is mediated by CYP3A in rat liver microsomes. Copyright © 2013 John Wiley & Sons, Ltd. Keywords: xylazine; ketamine; anesthesia; metabolism; microsomes; CYP3A; mass spectrometry; bioanalysis Introduction Xylazine, an a 2 -adrenoceptor agonist, is widely used in veteri- nary medicine in combination with ketamine for the anesthe- sia of rodents and rabbits (Stokes et al., 2009). Ketamine, an N-methyl-D-aspartate antagonist blocking glutamaergic func- tions, is used as a general anesthetic agent (Kronenberg, 2002). The a 2 -adrenoceptor agonists such as xylazine are used for their analgesic, sedative and muscle relaxant properties (Salonen, 1992; Plumb, 1999). The a 2 -adrenoceptors are located at the pre-synaptic level and are involved in the release of nor- adrenalin, leading to neural depression of the central nervous system. A recent investigation of the effects of endotoxemia on the pharmacokinetics and pharmacodynamics of ketamine and xyla- zine anesthesia in Sprague–Dawley rats was performed and the results suggest that endotoxemia may alter xylazine clearance (Veilleux-Lemieux et al., 2012). It is well known that pyrogens may decrease the metabolism of drugs by their action on liver cytochrome P450 in all animal species, including rats (Ueyama et al., 2005). Interestingly, the results from this study suggest an important impairment of xylazine clearance but not ketamine following an intramuscular administration of ketamine (80 mg/kg) and xylazine (5mg/mL) (Veilleux-Lemieux et al., 2012). The impairment of xylazine clearance leads to a significant increase in drug exposure and may result in unwanted effects. Therefore, dose adjustments might be necessary when fever or endotoxemia are present in animals to obtain a more rapid recovery after anesthesia. Additionally, hepatic clearance and drug–drug interac- tions are another concern, but better knowledge of xylazine metabolism is required. There are very few studies on the metabolism of xylazine and none have adequately characterized xylazine metabolic stability and the potential interaction between xylazine and ketamine. Extensive hepatic metabolism of xylazine was reported and six metabolites were identified in rat urine following an unusual high dose and route of administration (i.e. 1g p.o.; Mutlib et al., 1992). Moreover, in this study, in vitro metabolism data are * Correspondence to: Francis Beaudry, Groupe de Recherche en Pharmacologie Animal du Québec (GREPAQ), Département de biomedicine vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada. Email: francis.beaudry@umontreal.ca a Groupe de Recherche en Pharmacologie Animal du Québec (GREPAQ), Département de biomedicine vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada b UMR 5280 CNRS Université de Lyon 1, Institut des Sciences Analytiques, Université de Lyon, 69622 Villeurbanne cedex, France c Département de biomedicine vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada Abbreviations used: CYP, cytochrome P450; EPI, enhanced product ion mode; SRM, selected reaction monitoring. Biomed. Chromatogr. 2013 Copyright © 2013 John Wiley & Sons, Ltd. Research article Received: 27 November 2012, Revised: 7 January 2013, Accepted: 8 January 2013 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bmc.2875