(+)-Catechin—laccase oxidation dimeric standards were hemi-synthesized using laccase from Trametes versicolor in a water-ethanol solution at pH 3.6. Eight fractions corresponding to eight potential oxidation dimeric products were detected. The fractions profiles were compared with profiles obtained with two other oxidoreductases: polyphenoloxidase extracted from grapes and laccase from Botrytis cinerea. The profiles were very similar, although some minor differences suggested possible dissimilarities in the reactivity of these enzymes. Five fractions were then isolated and analyzed by 1D and 2D NMR spectroscopy. The addition of traces of cadmium nitrate in the samples solubilized in acetone-d6 led to fully resolved NMR signals of phenolic protons, allowing the unambiguous structural determination of six reaction products, one of the fractions containing two enantiomers. These products can further be used as oxidation markers to investigate their presence and evolution in wine during winemaking and wine ageing.
Rationale
The analysis of tannins is currently usually done by liquid chromatography after the chemical depolymerization of tannins. However, one of the main limitations of this method is the difficulty in specifically detecting each constitutive tannin monomer in a complex matrix, as numerous compounds co‐elute with the monomers, thereby compromising the analysis.
Methods
The depolymerization of tannins under acidic conditions and in the presence of thioglycolic acid releases the various constitutive monomers, either as terminal units or as extension units. The tannin subunits were then quantified using extracted‐ion chromatogram (EIC) mass spectrometry, which required the determination of the ionization efficiency of the monomers. Thus, we used AgBF4‐assisted coupling for the hemisynthesis of the noncommercial epicatechin gallate dimer.
Results
The EIC showed that the derivatives of the extension units were twice as ionized as the terminal units. Unlike the UV chromatograms, this new EIC‐based method is more specific and accurate because it is not impacted by the co‐elution phenomenon. This result, when added to the linearity obtained on a large range of the calibration curves, allowed for quantification of tannin subunits from EIC in complex mixtures with less pretreatment of the samples.
Conclusions
Due to its specificity and sensitivity, this EIC‐based method represents a significant step toward improving the quantification of the tannin composition of samples. The results of this study should allow the oxidation markers to be quantified more accurately and taken into account, thereby providing a better indication of the actual tannin composition.
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