Applicatins of liquid chromatography-tandem mass spectrometry to wine analysis : targeted analysis and compound identification

[摘要] ENGLISH ABSTRACT: The wine industry is an important sector of agriculture and wine analysis forms the basis ofassessing compliance of its commodities with regulatory standards and research in this field.Liquid chromatography (LC) is extensively used for the determination of a wide range of nonvolatilewine components, but conventional detectors impose performance limitations on thetechnique that prevents its application to sophisticated analytical problems. In particular,conventional detectors for LC often lack the sensitivity and specificity for the determination ofmany wine compounds, especially trace level analytes, and furthermore, do not possessspectral capabilities for compound identification or structure elucidation. The hyphenation ofmass spectrometry (MS) to LC has led to the introduction of a range of detectors that confershigh levels of sensitivity and selectivity to the technique. In addition, a wide variety of MSarchitectures are available that are inherently suited for targeted analysis or structureelucidation studies.In this dissertation, the potential benefits of liquid chromatography – tandem quadrupole massspectrometry (LC-MS/MS) to solve analytical problems relevant to the wine industry areexplored. LC-MS/MS is a particularly versatile analytical technique because both massanalysers can be operated in full-spectrum mode or selected-ion monitoring, which, togetherwith optional fragmentation, gives rise to four modes of operation that may be used for highlyspecific and sensitive targeted analysis or spectral investigations.In multiple reaction monitoring (MRM) mode, both analysers are set at single ion frequenciesspecific for the compound under investigation and one or more of its product fragments,respectively. MRM mode is ideally suited for trace level analysis in complex mixtures, even incases where the target components are not resolved from interferences. In this study, MRMdetection was used to solve challenges relevant to the wine industry for the selectivequantitation of target analytes that could not be analysed by conventional LC methods. Theapplication of this approach for the analysis of natamycin, ethyl carbamate (EC) and 3-alkyl-2-methoxypyrazines (MPs) in wine is demonstrated.Natamycin is an antimicrobial preservative that is not permitted in wine in the European Union.A rapid and sensitive method for the determination of natamycin was developed, and has been used since 2009 to regulate this vitally important sector of the South African wine exportindustry.EC is a natural carcinogen that occurs at trace level amounts in alcoholic products. It also hasthe potential to accumulate in wines and can occur in very high concentrations in some fruitbrandies. The determination of EC is complicated by its physicochemical properties, andavailable analytical methods suffer from drawbacks such as the requirement for elaborateextraction procedures and high solvent consumption. A novel method for the determination ofEC in wines, fortified wines and spirits is described and it was applied to perform an audit of theSouth African industry as well as to investigate factors responsible for its accumulation inalcoholic beverages. This work forms an integral part of the food safety mandate of the Stateand it ensures that export products comply with international norms for trade.MPs are ultra-trace-level aroma compounds that contribute to the varietal character ofSauvignon blanc wines. Their analytical determination is challenging due to their low levels ofoccurrence. The loading capacity of LC combined with the sensitivity and resolving power of MSwas exploited to analyse concentrated extracts, in order to achieve very low limits of detection.The performance of the LC-MS/MS method enabled the quantitation of these compounds attheir natural levels of occurrence, including the first quantitation and spectral confirmation of 3-ethyl-2-methoxypyrazine in wine. Extensive data pertaining to South African Sauvignon blancwines are reported and statistical analysis is performed, reporting the correlation of variablessuch as vintage and origin as well as wine parameters such as malic acid with wine MPs.Furthermore, the application of LC-MS/MS for structural elucidation and screening of targetclasses of analytes was demonstrated for the analysis of red wine anthocyanins. Theanthocyanidin-glycosides are responsible for the colour of red grapes and wine, contribute tothe sensory properties of wine, and are also of interest due to their beneficial biologicalproperties. Their determination is complicated by their large numbers and structural diversity,further exacerbated by diverse reactions during wine ageing as well as the lack of referencestandards for most members of this class of compounds. Tandem MS in scan mode was usedfor the highly selective detection of glycosylated anthocyanins and derivatives, exploiting thepredictable elimination of the sugar moiety in neutral loss mode. Concurrent survey scanexperiments were used to unambiguously identify neutral loss detected compounds. Themethod therefore follows a simplified and structured approach for unambiguous peak identification based on elution order and mass spectral information to impart a high level ofcertainty in compound identification.In summary, the work presented in this dissertation demonstrates that LC-MS/MS is a versatileand powerful analytical approach for the analysis of diverse compounds of relevance to the wineindustry. The sensitivity and specificity of MRM mode, and the selectivity and spectralcapabilities of neutral loss and survey scan modes of MS/MS detection, is amply demonstratedby the applications presented in the dissertation.

[发布机构] Stellenbosch University