Copyright © 2006 John Wiley & Sons, Ltd. Biomed. Chromatogr. 20: 1206–1215 (2006) DOI: 10.1002/bmc 1206 Z. Wang et al. ORIGINAL RESEARCH ORIGINAL RESEARCH Copyright © 2006 John Wiley & Sons, Ltd. BIOMEDICAL CHROMATOGRAPHY Biomed. Chromatogr. 20: 1206–1215 (2006) Published online 23 June 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/bmc.686 Comparison of supercritical fluid chromatography and liquid chromatography for the separation of urinary metabolites of nobiletin with chiral and non-chiral stationary phases Zhenyu Wang, 1 * Shiming Li, 1 Malgorzata Jonca, 1 Ted Lambros, 1 Stephen Ferguson, 1 Robert Goodnow 1 and Chi-Tang Ho 2 1 Discovery Chemistry, Hoffmann-La Roche Inc. 340 Kingsland Street, Nutley, NJ 07110, USA 2 Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA Received 21 March 2006; revised 10 April 2006; accepted 11 April 2006 ABSTRACT: Nobiletin (NOB), a polymethoxylated flavone found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes, yielding multiple hydroxylated metabolites. However, it has not been possible to date to separate the two demethylated nobiletin metabolites, 3′-demethyl-NOB and 4′-demethyl-NOB (regio-isomers) on reversed-phase liquid chromatography (RPLC). Additionally, both display similar mass spectrometric fragmentation, resulting in difficulties to identify the dominant metabolite. A successful separation method was developed by utilizing supercritical fluid chro- matography (SFC) with chiral stationary phase. The separation was also attempted with normal-phase liquid chromatography (NPLC) in both chiral and non-chiral modes. Chromatographic separation for the two nobiletin metabolites was superior by SFC than by LC, especially using chiral stationary phase. By comparing the SFC profile of the synthesized standards, the major nobiletin metabolite in mouse urine was identified as 4′-demethyl-NOB, with the concentration of 28.9 μg/mL. Copyright © 2006 John Wiley & Sons, Ltd. KEYWORDS: chiral separation; normal phase separation; nobiletin; metabolite; supercritical fluid chromatography; liquid chromatography *Correspondence to: Z. Wang, Discovery Chemistry, Hoffmann-La Roche Inc. 340 Kingsland Street, Nutley, New Jersey 07110, USA. E-mail: zhenyu.wang@roche.com Abbreviations used: ALS, automatic liquid sampler; NOB, nobiletin; NPLC, normal-phase liquid chromatography; RPLC, reversed-phase liquid chromatography; SFC, supercritical fluid chromatography; TCM, thermal control module. INTRODUCTION Flavonoids, a class of chemically related polyphenols of plant origin, exist ubiquitously in nature and also exhibit a broad spectrum of pharmacological properties (Silalahi, 2002; Manthey et al., 2001; Middleton et al., 2000; Whitman et al., 2005). Flavonoids are some of the most common secondary metabolites of fruit, vegetables, nuts, wine and tea. The human dietary in- take of these natural products is estimated to be about 1 g/day of mixed flavonoids (Okuno and Miyazawa, 2004). Nobiletin [Fig. 1(a)], a polymethoxyflavonoid (5,6,7,8,3′,4′-hexamethoxyflavone) found in sweet orange (Citrus sinensis) peel, is currently recognized as a promising anti-inflammatory and anti-tumor agent. Nobiletin has been reported to exhibit antiproliferative activity toward a human squamous cell carcinoma cell line (Kandaswami et al., 1991) and inhibitory activity on HL-60 cell differentiation (Kawaii et al., 1999), to inhibit tyrosinase activity (Sasaki and Yoshizaki, 2002) and to exhibit antimutagenic activity (Kanazawa et al., 1995). It is believed that, by undergoing metabolic biotransformation in vivo, nobiletin is demethylated by hepatic P450 enzymes yielding multiple hydroxylated metabolites, primarily, 3′-demethylnobiletin and 4′- demethylnobiletin [Fig. 1(b) and (c)]. In this study, chromatographic efforts are discussed for the separa- tion of these two regio-isomers. A successful separation method was developed by utilizing supercritical fluid chromatography (SFC) with chiral stationary phase. The result was also compared with high-performance liquid chromatography (HPLC) under normal-phase mode by using chiral and non-chiral stationary phases. EXPERIMENTAL Instruments. HPLC was performed on Agilent 1100 LC system (Wilmington, DE, USA) equipped with a diode array detector. SFC was performed on Berger MiniGram