Eur Food Res Technol (2005) 221: 798–802 DOI 10.1007/s00217-005-0038-6 ORIGINAL PAPER David Mu ˜ noz Rodr´ ıguez · Katarzyna Wrobel · Kazimierz Wrobel Determination of aldehydes in tequila by high-performance liquid chromatography with 2,4-dinitrophenylhydrazine derivatization Received: 15 March 2005 / Revised: 12 May 2005 / Accepted: 20 May 2005 / Published online: 16 August 2005 C  Springer-Verlag 2005 Abstract In this work, the determination of aldehydes in different tequila brands was carried out by high- performance liquid chromatography after derivatization with 2,4-dinitrophenylhydrazine. For the comparative pur- poses, two commercial brandies were also analyzed. The derivatization agent (50 μl of 3.5 mmol l −1 DNPH in HCl, 2 mol l −1 ) was added directly to the sample (500 μl) and dinitrophenylhydrazones formed were extracted with hex- ane. After evaporation of the solvent in nitrogen stream, the residues were dissolved in 100 μl of acetonitrile. The calibration standards were prepared from respec- tive dinitrophenylhydrazones, following the same proce- dure as for beverage samples. Reversed phase chromato- graphic separation was achieved on Luna C18 column (250 mm×4.6 mm, 5 μm), using gradient elution (acetoni- trile:water, from 68 to 80% of acetonitrile in 20 min) and a total flow rate 1 ml min −1 . Spectrophotometric detection for furanic aldehydes was at 390 nm (for other aldehy- des at 365 nm). The assignation of chromatographic peaks was accomplished by comparison of their relative reten- tion times and UV/Vis spectra with those of external stan- dards. The method of standard addition was also used. The aldehydes identified were 5-hydroxymethylfurfuraldehyde (t ret =4.1 min), formaldehyde (t ret =5.1 min), syringalde- hyde (t ret =5.6 min), acetaldehyde (t ret =6.2 min), 2- furaldehyde (t ret =7.2 min) and 5-methylfurfuraldehyde (t ret =8.9 min). At least four chromatographic peaks with retention times higher than 12 min remained unidentified. The quantification results showed drastically higher con- centrations of 2-furaldehyde and 5-methylfuraldehyde in tequilas as referred to brandies. Furthermore, 100% tequi- las contained higher levels of these two compounds (for four brands analyzed, mean values 18.6 and 5.97 μg ml −1 , D. M. Rodr´ ıguez · K. Wrobel · K. Wrobel () Instituto de Investigaciones Cient´ ıficas, Universidad de Guanajuato, L de Retana No. 5, 36000 Guanajuato, Mexico e-mail: kazimier@quijote.ugto.mx Tel.: +52-4737327555 Fax: +52-4737326252 respectively) as compared to the mixed brands (five brands analyzed, mean values 6.46 and 3.30 μg ml −1 ). The re- sults obtained confirm that the profile of furanic aldehydes depends on the type of fructans contained in the raw ma- terial and also on heating treatment applied or not prior to fermentation. In contrast to other polysaccharides, in- ulin hydrolyzes at elevated temperature and the contribu- tion of Maillard browning reactions increases the produc- tion of furanic compounds. Our results indicate that the levels of 2-furaldehyde and 5-methylfuraldehyde could be used for discrimination between 100% and mixed tequila brands. Keywords Tequila . Aldehyde . High-performance liquid chromatography . Dinitrophenylhydrazine Introduction The identification and quantification of aldehydes in al- cohol beverages have often been undertaken [1–5]. The importance of these analyses is due to known toxic- ity of several aldehydes, including formaldehyde, ac- etaldehyde, acroleine, benzaldehyde and so on [6]. On the other hand, information on aldehydes profile may help in assessing the authenticity and/or aging condi- tions of different brands [3, 7–11]. Wooden barrels are typically used for aging liquors [12]. During heat treat- ment of the inner part of barrels, lignin contained in wood decomposes with formation of phenolic adehydes (vanillin, syringaldehyde, coniferaldehyde, sinapaldehyde) [3, 8]. To the lesser extent, furanic compounds such as 2-furaldehyde (2-FAL), 5-methylfurfuraldehyde (5-MFA) and 5-hydroxymethylfurfuraldehyde (5-HMF) are formed as the degradation products of cellulose and hemicellulose [5]. During aging process, aldehydes are extracted from wood to alcoholic beverage thus contributing in its final flavor [3, 8]. The important source of the furanic aldehydes in alcoholic beverages is the Maillard reaction, which oc- curs when sugars are heated above their melting point [3, 13].