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Approaches and Challenges for Analysis of Flavor and Fragrance Volatiles Yong Foo Wong and Philip J. Marriott* Australian Centre for Research on Separation Science, School of Chemistry, Monash University, Clayton, Victoria 3800, Australia chemical differences may result in subtle differences to their fragrance and flavor properties, which means that precise identification is mandatory if such properties are to be quantified. This includes volatiles derived from herbs and spices, fruit and other plant extracts, and transformed products resulting from processing conditions. The widely used technologies of GC and GC−MS and the reliance of analysts on the wealth of qualitative and quantitative data available from routine application of GC and GC−MS, however, does not recognize that GC−MS often lacks the ability to distinguish components with similar structure and/or mass spectra. Isomeric forms among various chemical classes of components in complex samples mean that the peak capacity of a column (how many compounds a column can resolve just to baseline) is often far exceeded by the number of components, especially when they cluster in a narrow elution range. In this case, peak overlap will be the general expectation. However, for accurate and reliable identification, each peak should ideally be wellatural flavors and fragrances are generally mixtures in a resolved and appear in the detector as a single component. background matrix, with many similar structures. As a While deconvolution is now a well-established procedure, single result of their physicochemical characteristics and a prevailing (pure) components guarantee the highest quality spectra and sensory response, these compounds are crucial to the food, valid comparison to reference spectra and avoid ambiguities essential oil, and cosmetic fields.1 The use of flavor and resulting from contributions of co-eluting components to an fragrance substances for enjoyment, religious, or medicinal unresolved peak.3 The most compromised situation is where a reasons is as old as mankind. Today, the production of flavors major component strongly overlaps a trace component. In this and fragrances has been transformed, evolving from physical case, the trace component might very well be “invisible” to the and chemical isolation from natural plant resources as in analysis; it will simply not be measured. In flavor and fragrance ancient times and still of immense importance today, to analysis, we are often more interested in the key minor chemical synthesis or biological (enzymatic or microbial) components that may be masked by major components. 2 processes to generate desired fragrance and flavor compounds. Through technological advances, the higher separation order The large number of fragrance and flavor compounds in natural GC technologies (i.e., multiple columns) of heart-cut multisamples with similar structures and physicochemical characterdimensional GC (GC−GC) and its more recent innovative istics, different polarities, and a wide range of volatilities analogue of comprehensive two-dimensional GC (GC × GC) demand the use of highly efficient separation techniques. represent quests for enhanced selectivity and specificity.4 These Developments in analytical technology for volatile compounds technologies lead to much greater “separation space”, with are critically relevant, because poor resolution of target volatiles considerably more components readily recognized in the from matrix compounds and the lack of precision in analysis, usually as single, resolved peaks. Analytically, identification that limits structural assessment of compounds elimination of underlying interferences should produce peaks in complex matrices raises challenges at the frontier of of higher purity with better baseline signals. However, MS may instrument capabilities. The difficulty is compounded at lower still fail to adequately identify compounds because of the compound abundance. Flavor/fragrance compounds are absence of the compounds in available databases or the lack of especially prone to poor specificity in mass spectrometry, specificity of spectra (e.g., for isomers or related compounds). arising from their many isomers. We refer to this as “MS information shortfall”. Ideally, the use The development of gas chromatography (GC) in the 1950s of authentic standards (when available) to give correspondence and then capillary GC with mass spectrometry (MS) detection of GC retention and MS spectrum for the target compound is considered one of the greatest enabling analytical increases certainty of accurate, confident, and rigorous technologies for chemical analysis of volatile and semivolatile identification, but use of standards may still be insufficient. flavor compounds. Many of these compounds are derived from natural materials; they possess considerable structural heterogeneity but often have similar molecular formulas (e.g., many Received: July 8, 2017 compounds are based on terpene-type structures). Minor
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DOI: 10.1021/acs.jafc.7b03112 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry
Figure 1. Flowchart illustrating GC−MS compound selection strategy, data processing, and compound identification, as partly expounded in refs 6 and 7. Filter criteria limits are not defined; analysts can delineate their required identification levels (minimum reporting standard guidelines, Metabolomics Standards Initiative).5 (∗) 2D retention indices are applicable to multidimensional gas chromatographic (MDGC and GC × GC) analysis, and (#) 2D chemical property map criteria are applicable to GC × GC analysis. b/gd = background.
standards are not available (such as for “omics”-based studies).6,7 This workflow underscores the importance of adequate “filtering processes” to reduce the prevalence of false identifications and employing further data interpretation steps, retention indices, accurate mass, and structure-retention knowledge (position in 2D space), to achieve improved identification. Heart-cut GC−GC−MS [targeted analysis but also untargeted for selected chromatographic regions of a onedimensional (1D) analysis] and GC−MS are still applicable in this strategy, but co-elution in the latter case still remains a concern. Proper identification is a universal requirement for almost all analysis tasks using chromatographic separations with MS. Through systematic and careful application of chromatographic and MS filtering processes, substantial reduction of ambiguity in compound identification is achievable. Of course, both positive identification and absolute quantitative measurement are still sought through use of authentic standards when available. If indeed we can succeed in improved compound characterization where authentic standards are not available, then knowledge of sample composition will benefit. In our opinion, such an approach could be applied to many classes of flavor and fragrance substances derived from natural plant resources. Analysts need to address the appropriateness of their analytical methods to ensure that they are able to adequately claim identification of components at a sufficient level of precision; tentative assignment of identity carries with it additional uncertainty of material characterization. The challenges of improving analytical separation, accompanied by improved identification, are continually evolving as new capabilities are introduced.
The cost of individual authentic standards is substantial when extensive libraries of standards are required (e.g., for metabolomics). The lack of valid authentication of identity is or should be a considerable source of concern in many laboratories. The use of GC × GC and/or multidimensional GC−MS data, supported by two-dimensional (2D) retention indices, mass fragmentation data, mass accuracy, and spatial distribution of compounds in the 2D space, can provide a higher degree of confidence for tentative identifications, may improve quantitative data by removing underlying peak overlaps, and lead to lower detection limits. Tentative identifications should be claimed when the degree of confidence is insufficient for absolute identification. Identification of flavor compounds by GC−MS analysis is often based on a comparison of acquired compound spectra to MS libraries and an assessment of compound retention indices with known reference values. However, it is important to note the likelihood for rejection of low abundance or co-eluting compounds, which normally provide lower MS matches. Higher threshold MS match scores cannot be guaranteed as correct. The use of high-resolution MS for exact mass analysis can provide increased confidence for further confirmation of heteroatomic components with different molecular formulas. Chromatographic resolution becomes important when MS is unable to provide sufficient selectivity to discriminate, for example, isomeric species (e.g., flavors and fragrances generally consist of various chemical classes of terpenes). Application of multidimensional GC separations provides two sets of retention information (i.e., retention indices in first and second dimensions) according to the phase selectivity in each dimension, adding greater informing power for identification. A proposed workflow strategy (Figure 1) for the untargeted analysis of plant-derived materials has considerable merit by combining 2D retention index information, accurate mass MS, and concepts of peak positions in 2D space (i.e., structured retention patterns) for GC × GC, particularly when authentic
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Philip J. Marriott: 0000-0001-5180-1041 B
DOI: 10.1021/acs.jafc.7b03112 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
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Journal of Agricultural and Food Chemistry Notes
The authors declare no competing financial interest.
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REFERENCES
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DOI: 10.1021/acs.jafc.7b03112 J. Agric. Food Chem. XXXX, XXX, XXX−XXX
