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, th e most abundant amino acid in wine, and with the relative isomeric pre sence in samples of different vintage and geographical origin. The sam ple preparation was carried out by using Marfey's reagent ((1-fluoro-2 ,4-dinitrophenyl)-5-L-alanine amide (FDAA)) for the derivatisation rea ction in order to obtain DL and LL diastereomers of the corresponding optically active amino acids. The separation was performed with an ana lytical column for RP-HPLC Nucleosil 100-5 C18 (25 x 0.4 cm id), adopt ing both isocratic and gradient procedures and making use of water/ace tonitrile buffers as the mobile phase. It has been observed that the b est resolution is achieved under gradient conditions, although the ana lysis takes a long time. L-Norleucine was used as the internal standar d for quantitative determination and the detection of components was m ade at the fixed wavelength of 340 nm. Peak identifications were perfo rmed by comparing retention times and observing on-line UV spectra. Un der these conditions the detection limit for proline isomers reached t he value of 0.5 mg liter(-1). Wine samples of different ages were anal ysed and it is possible to see that L-proline is present only in 'youn g' wines, while the D-isomer appears in wines that are at least 5 year s 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 rati o D to D + L.