Available online at www.sciencedirect.com Journal of Chromatography B, 861 (2008) 56–63 Development and validation of a chemical hydrolysis method for dextromethorphan and dextrophan determination in urine samples: Application to the assessment of CYP2D6 activity in fibromyalgia patients Y. Daali a,∗ , S. Cherkaoui b , F. Doffey-Lazeyras a , P. Dayer a , J.A. Desmeules a a Clinical Pharmacology and Toxicology, Geneva University Hospitals, CH-1211 Geneva 14, Switzerland b Bracco Research SA, 31 route de La Galaise, 1211 Plan-les-Ouates, Switzerland Received 11 July 2007; accepted 16 November 2007 Available online 23 November 2007 Abstract Dextromethorphan (DEM) is a widely used probe drug for human cytochrome P450 2D6 isozyme activity assessment by measuring the ratio between DEM and its N-demethylated metabolite dextrorphan (DOR). DOR is excreted in urine mainly conjugated to glucuronic acid. Prior to quantification, DOR must be deconjugated to avoid variability caused by the polymorphic glucuronosyltransferase enzyme. A chemical hydrolysis method was optimized using a chemometric approach. Three factors (acid concentration, hydrolysis time and temperature) were selected and simultaneously varied to study their effect on conjugated DOR hydrolysis. Hydrolysis conditions that maximize DOR release without significant degradation of both DEM and DOR were chosen and results were compared to those obtained by enzymatic method using -glucuronidase. An HPLC method with fluorescence detection was developed for the simultaneous quantitation of DEM, DOR and levallorphan, used as an internal standard. Separation was performed on a phenyl analytical column (150 mm × 4.6 mm i.d., 5 m) with a mobile phase consisting of 18% acetonitrile and 50 mM phosphoric acid (pH 3). The overall analytical procedure was validated and showed good performances in terms of selectivity, linearity, sensitivity, precision and accuracy. Finally, this assay was used to determine DEM/DOR molar ratios in fibromyalgia patients for the purpose of determining phenotype status for the CYP2D6. © 2007 Elsevier B.V. All rights reserved. Keywords: Chemical hydrolysis; Experimental design; CYP2D6; Phenotype; Dextromethorphan 1. Introduction Drug pharmacokinetics may vary considerably between indi- viduals, and may be caused by differences in expression and activity of cytochrome P450 enzymes [1]. For CYP2D6, more than 70 different allelic variants have been described [2]. Differ- ent phenotypes can be distinguished: poor metabolizers (PM) lack the functional enzyme; intermediate metabolizers (IM) carry two different alleles, leading to partial activity; efficient metabolizers (EM) have two normal alleles; and ultra-rapid metabolizers (UM) have multiple gene copies [3]. DEM is a widely used and validated probe for assessing CYP2D6 ∗ Corresponding author. Tel.: +41 22 379 54 30; fax: +41 22 382 99 40. E-mail address: youssef.Daali@hcuge.ch (Y. Daali). activity [4–7]. DEM is metabolized to DOR by CYP2D6, and to 3-methoxymorphinan (3-MM) by CYP3A4 (Fig. 1). Moreover, 3-hydroxymorphinan (3-OH) is obtained through N,O-didemethylation by CYP3A4 and CYP2D6, respectively [8]. DOR is excreted in urine mainly conjugated to glucuronic acid [9]. Prior to quantification, urine must be deconjugated to avoid variability caused by the polymorphic glucuronosyl- transferase enzyme. Hydrolysis of conjugated DOR is generally achieved by enzymatic method using -glucuronidase [10]. However, this method is expensive, tedious and time consuming (18–24 h). A new chemical hydrolysis method using hydrochlo- ric acid is described in this study. The most important parameters are hydrolysis time, temperature and acid concentration. Reac- tion optimization is achieved by using a chemometric approach which represents a valuable statistical tool for the development 1570-0232/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2007.11.019