Separation of oxazepam, lorazepam, and temazepam enantiomers by HPLC on a derivatized cyclodextrin-bonded phase: application to the determination of oxazepam in plasma Chuong Pham-Huy a, * , Ge ´raldine Villain-Pautet a , He Hua a,b,c , Nassima Chikhi-Chorfi a , Herve ´ Galons d , Marc Thevenin a , Jean-Roger Claude a , Jean-Michel Warnet a a Laboratory of Toxicology, Faculty of Pharmacy, University of Paris V, 4 avenue de l’Observatoire, 75006 Paris, France b Division of Analytical Chemistry, China Pharmaceutical University of Nanjing, 24 Tong Jia Xiang, 210009 Nanjing, PR China c School of Environmental Science, Nanjing University, 210093 Nanjing, PR China d Laboratory of Organic Chemistry, Faculty of Pharmacy, University of Paris V, 4 avenue de l’Observatoire, 75006 Paris, France Abstract The enantioselective high-performance liquid chromatography (HPLC) of three racemic 3- hydroxybenzodiazepines, oxazepam (Oxa), lorazepam (Lor), and temazepam (Tem), is a difficult operation because of the spontaneous chiral inversion in polar solvent. To solve this problem, we have developed an HPLC method based on a chiral Cyclobond I-2000 RSP column, maintained at 12 jC, and a reversed mobile phase (acetonitrile in 1% triethylamine acetate buffer, TEAA) at a flow rate of 0.4 ml/min. Peaks were detected by a photodiode-array detector at 230 nm for quantification and by an optical rotation detector for identification of (+) and () enantiomers. The results showed that peak resolutions of Oxa, Lor, and Tem enantiomers, analyzed under the same conditions, were 3.2, 2.0, and 1.8, respectively. For the determination of Oxa enantiomers in plasma of rabbits, extraction with diethyl ether at pH 1.5, a polar organic mobile phase, and a Cyclobond I-2000 SP column were used. Other analytical conditions were the same as previously described. Blood samples were immediately cooled at 4 jC and centrifuged at 0 jC for the collection of plasma. The results showed a difference in plasma S(+)- and R()-oxazepam concentrations in rabbits. No 0165-022X/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII:S0165-022X(02)00123-9 * Corresponding author. Tel.: +33-1-5373-9835; fax: +33-1-4674-6644. E-mail address: pham.huy.chuong@wanadoo.fr (C. Pham-Huy). www.elsevier.com/locate/jbbm J. Biochem. Biophys. Methods 54 (2002) 287 – 299