J. Sep. Sci. 2007, 30, 669 – 672 R. Lopez et al. 669 Ricardo Lopez 1 Paola Dugo 2 Luigi Mondello 3 1 Department of Analytical Chemistry, Faculty of Sciences, Universidad de Zaragoza, Zaragoza, Spain 2 Dipartimento di Scienze degli Alimenti e dell'Ambiente, Facoltà di Scienze, Università di Messina, Messina, Italy 3 Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Messina, Italy Original Paper Determination of trans-resveratrol in wine by micro- HPLC with fluorescence detection A new method for the quantitative determination of trans-resveratrol in wine has been optimized and validated. The method is based on the direct injection of 500 nL of wine in an HPLC system fitted with an RP microcolumn of 10 cm60.32 mm and spectrofluorometric detection. The linear dynamic range of the method covers the normal range of occurrence of the analyte in wines and extends for two orders of magnitude with r 2 = 0.9994. Twenty-three wines have been analyzed with the pro- posed method, finding concentrations in the range reported by other authors. Keywords: Fluorescence / Micro-HPLC / trans-Resveratrol / Wine / Received: August 4, 2006; revised: November 8, 2006; accepted: November 9, 2006 DOI 10.1002/jssc.200600300 1 Introduction Grapes and wine contain large quantities of phenolic compounds of interest from a biochemical point of view. Among them, trans-resveratrol has attracted researchers because of its potential benefits on human health, which include anticarcinogen and antioxidant activities, plate- let aggregation inhibition, and lipidic metabolism mod- ulation [1]. trans-Resveratrol bioavailability is important owing to its reputation to benefit people suffering from cardiovascular diseases [2], the major dietary sources being peanuts, grapes, and wine. trans-Resveratrol con- tent depends on grape variety, with larger quantities in red wines than in white wines, regardless of the wine- making technology applied [3]. The health related properties of trans-resveratrol have led to a wide variety of analytical methods developed for its analysis in wines. The methods published comprise GC with mass spectrometric detection [4] or flame ioniza- tion detection [5], CE with fluorescence spectroscopy [6, 7], single wavelength [8], or diode array UV detection [9], but most of the methods are based on RP-HPLC. Detection under HPLC conditions has been carried out using UV absorption [10–13], electrochemical methods [14], MS [15, 16], MS/MS [17], and fluorimetric detection [18–22]. In the first method proposed, sample preparation steps were used, i. e., derivatization [23], liquid–liquid extrac- tion [20, 24], or SPE [4, 22], however, currently the trend is to use direct injection of the sample [10, 19, 25]. In recent years, interest in microcolumn LC (micro-LC) has increased considerably. This is mainly due to the abil- ity to work with small sample sizes, small volumetric flow rates, and enhanced detection performance with the use of concentration-sensitive detectors due to the reduced chromatography dilution [26, 27]. Furthermore, the very small flows used in micro-LC allow direct cou- pling with MS detectors, and facilitate coupling with multidimensional LC systems [28]. The aim of the present work was the development and validation of a micro-LC method for the quantitative determination of trans- resveratrol in the concentration range usually found in wines. 2 Experimental 2.1 Reagents Water (Prolabo, Fontenay sous Bois, France), ACN (BDH, Poole, England), and methanol (J. T. Baker, Deventer, The Netherlands) were of HPLC grade. Acetic acid was from Carlo Erba (Rodano, Italy). trans-Resveratrol was pur- chased from Sigma (Steinheim, Germany). A stock solu- tion (1000 mg/L) of trans-resveratrol in methanol was pre- pared and stored at –208C. Diluted solutions to identify trans-resveratrol and to build the calibration graph were prepared just before analysis. All the solutions were shielded from light. 2.2 Apparatus The HPLC analyses were performed on an HPLC system equipped with an RF-10AXL spectrofluorometric detector fitted with a microflow cell. Data were acquired and pro- cessed using Shimadzu CLASS-VP software. To reduce the Correspondence: Dr. Ricardo Lopez, Pedro Cerbuna 12, Facultad de Ciencias (Edif. D), 50009 Zaragoza, Spain E-mail: riclopez@unizar.es Fax: +34-976761292 Abbreviation: micro-LC, microcolumn LC i 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com