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

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 &
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