HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY DETERMINATION OF PROLINE ISOMERS IN ITALIAN WINES

This paper deals with the quantitative reverse-phase high-performance liquid chromatography (RP-HPLC) analysis of the isomers of proline, the most abundant amino acid in wine, and with the relative isomeric presence in samples of different vintage and geographical origin. The sample preparation was carried out by using Marfey's reagent ((1-fluoro-2,4-dinitrophenyl)-5-L-alanine amide (FDAA)) for the derivatisation reaction in order to obtain DL and LL diastereomers of the corresponding optically active amino acids. The separation was performed with an analytical column for RP-HPLC Nucleosil 100-5 C18 (25 x 0.4 cm id), adopting both isocratic and gradient procedures and making use of water/acetonitrile buffers as the mobile phase. It has been observed that the best resolution is achieved under gradient conditions, although the analysis takes a long time. L-Norleucine was used as the internal standard for quantitative determination and the detection of components was made at the fixed wavelength of 340 nm. Peak identifications were performed by comparing retention times and observing on-line UV spectra. Under these conditions the detection limit for proline isomers reached the value of 0.5 mg liter(-1). Wine samples of different ages were analysed and it is possible to see that L-proline is present only in 'young' wines, while the D-isomer appears in wines that are at least 5 years old. The most abundant D-proline content was observed in a 30 years vintage red wine of Puglie (southern Italy) with about 15% of the ratio D to D + L.