Development of a novel LC-MS/MS method for the detection of adulteration of South African sauvignon blanc wines with 3-alkyl-2-methoxypyrazines
A method for the detection of adulteration of South African Sauvignon blanc wines, by enrichment with foreign sources of 3-alkyl-2-methoxypyrazenes, is described. The levels of three 3-alkyl-2-methoxypyrazenes (3-isobutyl-, 3- isopropyl- and 3-sec-butyl-2-methoxypyrazine) in South African Sauvignon blanc wines were measured with liquid chromatography-mass spectrometry. Sample preparation involved clean-up and pre-concentration by distillation followed by solvent extraction of the distillate with dichloromethane. Extracts were acidified and concentrated by evaporation and finally reconstituted to a fixed volume to affect quantitative pre-concentration of the samples. Sample extracts were separated with reversed phase liquid chromatography utilizing a phenyl-hexyl separation column. Residues were measured with liquid chromatography-mass spectrometry utilizing a tandem quadrupole mass spectrometric detector operated in multiple reaction monitoring mode for optimal trace level quantitation. Atmospheric pressure chemical ionization was utilized as electrospray ionization was found to suffer from quenching effects attributed to the sample matrix. Qualitative information was obtained from the relevant molecular ions as well as two secondary ion transitions (and one ion ratio) in each case. Recoveries obtained by the extraction procedure were better than 90% with coefficient of variance of better than 10% at concentrations from 1 to 100 ng/L. The limit of detection of the method was 0.03 ng/L and the limit of quantification 0.10 ng/L for the three analytes measured. The described LC-MS method is more sensitive for the determination of 3-alkyl-2-methoxypyrazines in wine than GC methods reported for the same purpose. From the experimental data, a set of parameters were established to discriminate adulterated South African Sauvignon blanc wines. It was demonstrated that the 3-isobutyl-2-methoxypyrazine concentration, despite showing considerable variance, was confined to a relatively narrow range spanning approximately two orders of magnitude (0.20 to 22 ng/L). A clear indication of possible maximum values for this compound in South African Sauvignon blanc wines was obtained from the analysis of a large number of samples (577), spanning most relevant wine producing regions and representing vintages 2003 to 2006. It was also demonstrated that South African Sauvignon blanc wines contain the major 3- alkyl-2-methoxypyrazenes in reasonably distinct relative amounts and that the said ratios of abundance may be used to elucidate authenticity. The expected effect of adulteration with green pepper extracts or some synthetic preparations on the 3-isobutyl-2-methoxypyrazine concentration as well as the relative abundances were also determined by characterizing the corresponding profiles in green peppers and some synthetic flavor preparations. Two adulterated samples in the dataset were identified by both outlined criteria. A limited number of wines of other cultivars were also analyzed. The results represent the most complete and accurate data on the 3-alkyl-2-methoxypyrazine content of South African Sauvignon blanc wines to date. A publication covering the work presented in this thesis is currently in preparation.