Journal of Chromatography A, 868 (2000) 295–303 www.elsevier.com / locate / chroma Simultaneous stereoselective analysis of tramadol and its main phase I metabolites by on-line capillary zone electrophoresis–electrospray ionization mass spectrometry a a b c a, * Serge Rudaz , Samir Cherkaoui , Pierre Dayer , Salvatore Fanali , Jean-Luc Veuthey a Laboratory of Pharmaceutical Analytical Chemistry, University of Geneva, 20 Bd. d‘ Yvoy, 1211 Geneva 4, Switzerland b Clinical Pharmacology Division, University Hospital, Geneva 4, Switzerland c Istituto di Cromatografia del C.N.R., Monterontondo Scalo, Rome, Italy Received 1 September 1999; received in revised form 23 November 1999; accepted 23 November 1999 Abstract On-line combination of partial filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the simultaneous enantioseparation of tramadol and its main phase I metabolites. The partial filling technique was efficient at avoiding MS contamination by the chiral selector. Different experimental factors were investigated, including the chiral selector nature and concentration, plug length as well as the separation temperature. The best enantioseparation of the investigated compounds was achieved with a coated polyvinyl alcohol capillary and a 40 mM ammonium acetate buffer, pH 4.0, adding sulfobutyl ether b-cyclodextrin (2.5 mg / ml) as the chiral selector. The charged cyclodextrin not only allowed enantioseparation of tramadol and its metabolites, but also improved the selectivity of compounds with the same molecular mass. Finally, CE–electrospray ionisation-MS was successfully applied to the stereoselective analysis of tramadol and its main metabolites in plasma after a simple liquid–liquid extraction.  2000 Elsevier Science B.V. All rights reserved. Keywords: Enantiomer separation; Buffer composition; Partial filling capillary electrophoresis; Tramadol; Cyclodextrins 1. Introduction activity on the monoaminergic system [3]. Tramadol is formulated as a racemic mixture where each ( 1 / 2)- trans - 2 - [(Dimethylamino)methyl] - 1 - (3 - enantiomer displays different binding properties for methoxyphenyl)cyclohexanol hydrochloride various receptors [4–6]. (tramadol) is a centrally acting drug which does not After oral administration, tramadol is rapidly possess a pronounced opioid side-effect profile [1,2] absorbed and reaches peak plasma concentration in a and may exert part of its analgesic effect through mean of 2 h. About 85% of a tramadol dose is metabolized by the liver [4,7,8]. As shown in Fig. 1, tramadol undergoes biotransformation in the liver *Corresponding author. Tel.: 141-22-702-6336; fax: 141-22-781- through two main metabolic pathways to form N- 5193. E-mail address: jean-luc.veuthey@pharm.unige.ch (J.-L. Veuthey) and O-demethylated compounds (phase I reactions) 0021-9673 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0021-9673(99)01257-1