Determination of aldehydes in tequila by high-performance liquid chromatography with 2,4-dinitrophenylhydrazine derivatization

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 purposes, 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 hexane. After evaporation of the solvent in nitrogen stream, the residues were dissolved in 100 μl of acetonitrile. The calibration standards were prepared from respective dinitrophenylhydrazones, following the same procedure as for beverage samples. Reversed phase chromatographic separation was achieved on Luna C18 column (250 mm×4.6 mm, 5 μm), using gradient elution (acetonitrile: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 aldehydes at 365 nm). The assignation of chromatographic peaks was accomplished by comparison of their relative retention times and UV/Vis spectra with those of external standards. 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), syringaldehyde (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 concentrations of 2-furaldehyde and 5-methylfuraldehyde in tequilas as referred to brandies. Furthermore, 100% tequilas contained higher levels of these two compounds (for four brands analyzed, mean values 18.6 and 5.97 μg ml⁻¹, respectively) as compared to the mixed brands (five brands analyzed, mean values 6.46 and 3.30 μg ml⁻¹). The results obtained confirm that the profile of furanic aldehydes depends on the type of fructans contained in the raw material and also on heating treatment applied or not prior to fermentation. In contrast to other polysaccharides, inulin hydrolyzes at elevated temperature and the contribution of Maillard browning reactions increases the production 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.