Development and Application of An Integrated GC-MS/MS and GC-IRMS Analytical Approach in Food Quality Evaluation

Consumers, particularly those in developed countries, are becoming increasingly concerned not only about the composition of food products but also about their authenticity and geographical origin. Globalisation of food markets and the growing demand for high-quality natural ingredients have increased the economic value of many food products and flavourings. As a consequence, fraudulent practices such as adulteration, substitution, dilution, and mislabelling have become more frequent. Reliable identification of authentic products and detection of fraudulent ones have therefore become important challenges for food control authorities, producers, and consumers. In this context, it is of increasing importance to develop robust analytical approaches capable of verifying the authenticity and origin of food products. Plant extracts and essential oils represent a particularly vulnerable category of products in terms of food fraud. These materials are widely used in the food, cosmetic, pharmaceutical, and fragrance industries and often command high prices, especially when derived from natural botanical sources. Chemically, plant extracts and essential oils are complex mixtures containing hundreds of constituents with different chemical structures and concentrations. Among these compounds, volatile organic compounds (VOCs) play a fundamental role in defining the characteristic aroma and flavour profiles of plants. Because of their economic value and chemical complexity, plant-derived flavourings are frequently subject to adulteration with cheaper synthetic compounds or with materials of different botanical or geographical origin. Therefore, analytical approaches capable of simultaneously characterising chemical composition and providing information about the origin of compounds are essential for the authentication and evaluation of these products. Gas chromatography coupled with tandem mass spectrometry (GC-MS/MS) is widely used for the analysis of VOCs in plant extracts and essential oils. This highly sensitive and selective technique enables detailed compositional profiling of complex botanical matrices. Through chromatographic separation and mass spectrometric detection, GC-MS/MS allows the simultaneous identification and quantification of numerous compounds within a single analytical run and facilitates the identification of key molecules associated with specific plant materials. However, compositional analysis alone often provides limited information regarding the origin of the detected compounds, as natural and synthetic molecules may exhibit identical chemical structures and similar chromatographic behaviour. For this reason, additional analytical approaches are often required to provide complementary information that can support authenticity assessment and traceability determination. Compound-specific isotope analysis (CSIA) overcomes this limitation by enabling the determination of isotope ratios for individual molecules within a mixture. By combining chromatographic separation with isotope ratio mass spectrometry, CSIA provides detailed isotopic information at the molecular level and allows the investigation of biosynthetic pathways and production processes of target molecules. Despite its considerable potential, the application of CSIA to plant-derived flavourings remains relatively limited, and in many cases isotopic investigations have focused only on one or two major compounds. In addition, isotopic analysis is often performed independently from detailed compositional characterisation, which may limit the interpretation of the results. In this work, a gas chromatography-based approach integrating compositional profiling by gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) with compound-specific isotope ratio analysis (δ13C and δ2H) by gas chromatography-combustion/pyrolysis isotope ratio mass spectrometry (GC-C/Py-IRMS) was developed and applied to the characterisation and evaluation of herbs and flavouring materials. Within this integrated analytical strategy, GC-MS/MS was used to identify and quantify volatile compounds and to select suitable molecular targets for isotope analysis, while GC-IRMS provided compound-specific isotopic information that reflects their biosynthetic origin and production pathways. The combination of these two complementary analytical techniques makes it possible to obtain simultaneously both compositional and isotopic information for the same set of compounds, thereby providing a more comprehensive characterisation of complex botanical matrices. Two representative case studies were investigated in order to demonstrate the potential of this integrated approach: vanilla extracts and peppermint-flavoured products. These two flavouring materials were selected because of their wide use in various products and frequent involvement in cases of adulteration and mislabelling. The first case study focused on vanilla extracts, one of the most widely used natural flavourings in the food industry. Most previous studies addressing the authentication of vanilla products have concentrated primarily on vanillin, the main compound responsible for the characteristic aroma of vanilla. However, vanilla extracts contain numerous other aromatic constituents that may also provide valuable information for authenticity assessment. In this work, the analytical investigation was therefore extended beyond vanillin to explore additional molecular targets that could contribute to the authentication and traceability of vanilla-derived flavourings. Using GC-MS/MS compositional profiling, several minor aromatic compounds present in authentic vanilla pods were identified. Among these compounds, homovanillic acid, vanillyl alcohol, and 4-ethoxymethylphenol were identified as promising candidates for compound-specific isotope analysis. In parallel, the possible presence of ethyl vanillin or its potential precursors in natural vanilla extracts was also investigated, as ethyl vanillin is commonly used as a flavour enhancer in food products and may be added fraudulently to vanilla preparations. The results confirmed the absence of ethyl vanillin in authentic vanilla pods and demonstrated that, when chromatographically separated, ethyl vanillin does not interfere with the determination of vanillin isotope ratios by GC-IRMS. Importantly, this study represents the first application of compound-specific isotope analysis to minor aromatic compounds in vanilla pods, thereby extending the analytical framework available for the authentication of vanilla flavourings. The second case study addressed peppermint essential oil, a flavouring widely used in the food and beverage, cosmetic, pharmaceutical, and tobacco industries. Although numerous studies have investigated the volatile composition of peppermint extracts and essential oils, compositional analysis alone often provides limited information for the evaluation of product quality. In this study, the integrated GC-MS/MS and GC-IRMS workflow was applied to peppermint essential oils and to a variety of commercial mint-flavoured products. Using GC-MS/MS, more than forty volatile compounds were identified and quantified in peppermint samples, with menthol and menthone representing the predominant constituents. Compound-specific isotope ratios (δ13C and δ2H) were subsequently determined for selected target compounds using GC-IRMS. The analytical strategy was further extended to the investigation of commercial mint-flavoured products, including syrups, mouthwashes, candies, and chewing gums. By combining compositional and isotopic data, differences in both volatile profiles and isotope ratios were observed, providing complementary information for the comprehensive characterisation and quality evaluation of peppermint-flavoured products. Overall, the results obtained in this work demonstrate that the integration of compositional information obtained by GC-MS/MS with compound-specific isotopic data obtained by GC-IRMS provides a powerful analytical strategy for the characterisation of plant-derived flavourings. By simultaneously considering chemical composition and isotopic signatures, the proposed workflow allows a more detailed investigation of complex botanical matrices and improves the reliability of authenticity and quality assessments. The multi-analytical approach developed in this thesis therefore represents a valuable contribution to the field of food quality control and highlights the potential of combining compositional and isotopic techniques for the investigation of high-value natural products.