1. Figure 3 : Chomatographic separation of L(+) and meso trans-caftaric acid Redox Analysis of grape hydroxycinnamic acids: a new standardized quantification approach based on DIastereomeric Dilution Assay (DIDA) Aurélie Roland a*, Stéphane Delpech a, Anaïs Bouisseau b, Florine Cavelier b and Rémi Schneider a a Nyseos, 53 rue Claude François, Parc 2000, 34080 Montpellier, France b Institut des Biomolécules Max Mousseron - UMR 5247 CNRS-UM-ENSCM, Département des Aminoacides, Peptides et Protéines (DAPP), Université Montpellier, 34095 Montpellier cedex 05, France. *aurelie@nyseos.fr Introduction The hydroxycinnamoyltartaric esters or hydroxycinnamic acids (HA) (Figure 1) of grapes have been widely studied over the past thirty years. Trans-caftaric, fertaric and coutaric acids are of particular interest due to their high abundance and important role in several oxidation processes. Amongst the most important of those processes is the polyphenoxidase mediated formation of the Grape Reaction Product (GRP) from caftaric acid and glutathione. Whilst the process is very well understood mechanistically (1), quantitative analysis of the different HA ratios in grape musts remains a problem as they are not commercially available. The aim of our work was to develop a standardized analysis of HA and GRP in grapes, musts and wines by nanoChipCUBE-LC-MS/MS and DIastereomeric Dilution Assay (DIDA). Materials and methods For this purpose, we firstly developed the total synthesis of natural and diastereoisomeric analogues of HA and GRP by taking advantage of silicium chemistry (2) (Figure 2). These analytical standards have then been used to set up a standardized analysis of oxidation markers by DIastereomeric Dilution Assay (DIDA) (3) which conferred high accuracy and repeatability. Briefly, an analytical volume of sample was diluted in model must or wine and then analyzed in Multiple Reaction Monitoring mode under positive electrospray ionization. Chromatographic separation of natural analyte from its corresponding diastereoisomer required an important gradient optimization to reach a peak resolution at least equal to 2 (Figure 3). The full validation according to the OIV recommendations showed the high sensitivity (LOD 0.1 mg/L), repeatability and reproductibility (RSD < 6 %) and accuracy (86-103%) of the method for various enological matrices (grapes, musts, wines). In Vino Analytica Scientia 2015 - Trento, ITALY Results and Discussion This method has been applied for the analysis of 16 pilot scale musts of Merlot (2014) obtained according to 2 different preparation: -Classic: no warmed maceration -MPC: Merlot grapes have been destemmed then macerated under warmed conditions (75°C during 5h). MPC musts exhibited higher levels of HA and GRP than classic ones. So, warming conditions favoured the HA extraction from grapes since they are preferentially located into the berry skin. The classic must elaboration protocol conducted to most GRP production compared to MPC one. Indeed, until 50% of initial caftaric acid has been converted to GRP. Conclusion We proposed for the first time a new standardized method to quantify HA and GRP in enological matrices. HA are particularly reactive from chemical point of view and required a very accurate standardization. The use of meso-HA as internal standards (DIDA quantification) allowed very satisfactory analytical performances. Bibliography 1. Cheynier, V. F.; Van Hulst, M. W. J., Oxidation of trans-caftaric acid and 2-S-glutathionylcaftaric acid in model solutions. Journal of Agricultural and Food Chemistry 1988, 36, 10-15. 2. Bouisseau, A.; Cavelier, F.; Martinez, J.; Schneider, R.; Vialaret, A. Procédé d'estérification de molécules polaires, Patent, PCT/EP2014/065926. 24/07/2014, 2014. 3. Schlatterer, J.; Maurer, S.; Breithaupt, D. E., Quantification of 3R,3 ' R-zeaxanthin in plant derived food by a diastereomeric dilution assay applying chiral high-performance liquid chromatography. Journal of Chromatography A 2006, 1137, 216-222. Figure 2 : Patented chemical synthesis of natural and non-natural HA (2) Redox Acknowledgements We thanked the FEDER and the Languedoc-Roussillon Region for financial support. Figure 4: average concentration in Merlot musts L(+)- trans-caftaric acid Meso- trans-caftaric acid (ISTD) Figure 1 : Chemical structures of natural (L+) and non-natural (meso) HA Redox R = OH => trans-caftaric acid R = H => trans-coutaric acid R = Ome => trans-fertaric acid