title: Advanced gas chromatography with mass spectrometry for phytoanalysis of hop (Humulus lupulus L.)
Creators: name: Yan, D
Publisher: name: University of Tasmania
Year: 2019

description: The phytochemical composition of hop (Humulus lupulus L.) makes it a vital raw material for the brewing industry. However, the separation, detection, and identification of all components present within hops remains a major challenge. This thesis focuses on a range of developments and applications of advanced gas chromatography (GC) techniques, hyphenated with high resolution mass spectrometry (MS) for untargeted and/or targeted profiling of hop secondary metabolites in H. lupulus L. essential oils. One-dimensional gas chromatography (1D GC) is still the mainstay analytical method in plant essential oil analyses. Phytochemical characterisation of essential oils derived from 30 representative Australian hop genotypes, including experimental hybrid genotypes and commercial cultivars, using high resolution GC coupled with quadrupole accurate mass time-of-flight MS (Q-TOFMS) is described. The diversity of volatile phytoconstituents among the analysed samples was critically evaluated and interpreted in terms of their genetic and biogeographical origins, and in light of an understanding of the biosynthetic processes that result in accumulation of flavourrelevant metabolites in mature hop cones. This study highlights the potential and indicates the limitations of one-dimensional GC analysis of complex plant samples. The potential of comprehensive two-dimensional gas chromatography (GC×GC) combined with Q-TOFMS, to perform the untargeted profiling of essential oils derived from a representative range of hop genotypes, is discussed. Comprehensive overview and distinct differences of metabolic profiles were readily observed (based on 2D or 3D chromatograms) due to the orderly distributions of metabolites in the 2D separation space, allowing simple and fast fingerprinting of different hop genotypes. Distinguishable chemotype patterns were displayed among experimental and commercial hop genotypes, indicating genetic diversity among the samples studied. A complex array of secondary metabolites was detected, and oxygenated sesquiterpenes were proposed as a key chemical identifier group for different genotypes. The markedly different metabolite profiles were further interpreted through chemometric analysis, which allow the classification of three major chemotypes among analysed genotypes. The findings indicate that this high resolution chromatographic approach has great potential towards improved methodologies for better understanding of hop chemistry. Next, development of a four-column multiplexed technique with two independent twodimensional column ensembles (2GC×2GC), employing an additional gas supply and electronic pressure control at the midpoint between the two dimensions, is outlined. Appropriate carrier-gas flow control at the junction of the first-dimension (\(^1\)D) and second-dimension (\(^2\)D) columns permits the possibility and simplicity of implementing GC×GC and 2GC×2GC experiments. Comparison of analyses with and without independent \(^2\)D flow control, and the importance of applying flow control to adjust the separation speed in \(^2\)D were outlined. The analytical performance of the proposed multiplexed approach was demonstrated by the analyses of hop essential oil with two different column combinations. The unique ability to design two entirely independent conventional GC×GC separations for each injection, yielding two independent GC×GC chromatograms viewed in a single window leading to an appreciable gain of productivity was demonstrated. Lastly, a novel sequential three-dimensional GC hyphenated with Q-TOFMS (3D GC–Q-TOFMS) approach for profiling of secondary metabolites in complex plant extracts is described. The integrated system incorporates a preliminary non-polar first-dimension (\(^1\)D\(_{np}\)) separation step, prior to a microfluidic heart-cutting (H/C) of a targeted region(s) into a polar second-dimension (\(^2\)D\(_{PEG}\)) column for multidimensional separation (GC\(_{np}\)–GC\(_{PEG}\)). For additional separation, the effluent from \(^2\)D\(_{PEG}\) can then be modulated according to a GC×GC process, using an ionic liquid phase as a third-dimension (\(^3\)D\(_{IL}\)) column, to produce a sequential GC\(_{np}\)–GC\(_{PEG}\)×GC\(_{IL}\) separation. The described integrated system can be used in a number of modes, but one useful approach is to target specific classes of compounds for improved resolution. The analytical performance and applicability of the proposed approach is demonstrated and discussed through the separation and detection of the oxygenated sesquiterpenes in hop essential oil and agarwood (Aquilaria malaccensis) oleoresin. Improved resolution and peak capacity is illustrated through the progressive comparison of the tentatively identified components for GC\(_{np}\)–GC\(_{PEG}\) and GC\(_{np}\)–GC\(_{PEG}\)×GC\(_{IL}\) methods. The described methodology should be a valuable adjunct for the improved characterisation of complex plant samples, particularly in the area of plant metabolomics.