Identification of the major drivers of 'phenolic' taste in ... - GWRDC

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AWRI: Identification Of The Major Drivers Of ‘Phenolic’ Taste In White Wines Identification of Chromatography Peaks Identification was carried out initially by analysing commercially-produced HPLC-grade reference compounds by HPLC-DAD. Table A-2 lists obtained retention times (RT), UV detector response factors and spectral characteristics (max listed first) in the order they elute. A composite sample of Chardonnay, Riesling and Viognier wine made from whole bunch pressings, hard pressings and juice macerated for 60 hours was concentrated (wine freeze dried and reconstituted in 2% formic acid to yield a 10x concentration factor) was also analysed at the same time and this chromatogram and Figure A-4 : Reference compound HPLC-DAD chromatograms (graphically normalised) overlayed on a chromatogram of a mastermix of CHA, RIE, VIO whole bunch-pressed and high phenolic wines represents this chromatogram positioned under the peaks for the reference compounds. Comparing the RT of peaks in an unknown wine samples with this database of RTs and spectral characteristics it is possible to tentatively identify compounds in the unknown. However, there are potentially 100 phenolic compounds in white wine and the reference compounds cannot reflect all of these potential components. The only way to identify the larger majority of these compounds is to use an accurate mass detection system instead of the diode array UV detector. The technique of HPLC-ESI-QTOF-MS/MS (Electrospray ionization quadrupole time-of-flight mass spectrometry) allows the effluent of the chromatographic column to be sprayed into a mass spectrometer where the mass of eluting compounds is determined with an accuracy of four decimal places. By comparing the mass of the majority ion of each compound and the breakdown products against all possible combinations of carbon, hydrogen, oxygen and sulfur it is possible to identify chromatographic peaks and most importantly for those which no commercial reference compounds are available. The difference between the measured mass and the theoretical mass of a tentatively identified compounds is , given in mDa; this must be < 3 for a reliable fit of structure. All of the hydroxycinnamic acids have potentially two positional isomers (trans or cis) which cannot be assigned by mass spectrometry, only by running reference compounds or using NMR can these configurations be assigned and in the case of the tartaric esters and GRP conjugates these are not commerically available and therefore requiring lengthy and complicated synthesis. The same sample composite sample and reference compounds were analysed under the same chromatographic conditions as above. Trifluroacetic acid (TFA) was not included in the mobile phase to minimise ionisation suppression. This only had a marginal effect on peak shape for a small number of compounds. Major peaks were identified by comparing the exact mass of extracted molecular features against the MassBank database. With these new compounds identified, the UV spectra of the hitherto unknown peaks were added to the UV library potentially allowing better identification when using only the HPLC-DAD technique. A.3.4 Conclusion This new analytical method using two inline Gemini C6-phenyl HPLC columns now makes it possible to analyse greater than 80 identified phenolic components. This degree of separation has been achieved with only very minimal sample handling (removal of alcohol). Such degree of separation has only been achieved to date with extensive time-consuming extraction/isolation (Baderschneider & 110
AWRI: Identification Of The Major Drivers Of ‘Phenolic’ Taste In White Wines Winterhalter, 2000, 2001). A large proportion of these compounds are identified as glycosidically bound as well as methyl and ethyl esters of many of the phenolic acids. 350 10 20 30 40 50 60 70 80 90 300 250 200 150 100 50 0 Figure A-4 : Reference compound HPLC-DAD chromatograms (graphically normalised) overlayed on a chromatogram of a mastermix of CHA, RIE, VIO whole bunch-pressed and high phenolic wines 111 Gallic acid cis-Caftaric acid p-Hydroxybenzoic acid Tyrosol Gentisic acid tr-Caftaric acid Epigallocatechin Catechin Vanillic acid Caffeic acid Dihydroquercetin Sinapic acid Epicatechingallate Protocatachuic acid ethyl ester 10 20 30 40 50 60 70 80 90 350 300 250 200 150 100 50 0 Quercetin-3-glucoside Quericetin-O-3-rutinosde Quercetin -3-galactoside Quercetin -3-glucuronide Resveratrol Myricetin Syringetin-O-3-glucoside Fisetin Caffeic acid ethyl ester Chlorogenic acid Syringic acid Procyanidin B2 dimer Epicatechin Ethyl gallate p-Coumaric acid 96 98 102 104 106 96 98 100 102 104 106min Quercetin dihydrate Laricitrin Luteolin Epigallocatechin gallate Ferulic acid ethyl ester Kaempferol Isorhamnetin Apigenin Ferulic acid Syringetin
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Identification of the major drivers of 'phenolic' taste in ... - GWRDC

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