Identification of reaction products of caftaric acid and bisulfite present in commercial wines using high resolution mass spectrometric and nuclear magnet resonance techniques Yoji Hayasaka1, Cory Black1, Jeremy Hack1,2, Paul Smith1 1 The Australian Wine Research Institute, PO Box 197, Glen Osmond (Adelaide) SA 5064, Australia 2 SA Metabolomics Facility, PO Box 197, Glen Osmond (Adelaide) SA 5064, Australia Corresponding author’s email: [email protected] O COOH HO COOH (E)-caftaric acid O The questions: OH HO Oxidation • Does caftaric acid react with bisulfite? O COOH • Do the reaction products of caftaric acid and bisulfite exist in commercial wines? O COOH (E)-caftaric acid-O-quinones O O OH Reasons behind the questions: + + NH O 2 H N HSO3¯ • Bisulfite and glutathione are strong nucleophilic compounds (electron donors) and act as antioxidants in HOOC N COOH H bisulfite ion O wine HS Glutathione (GSH) • Grape reaction product (GRP) can be formed by a nucleophilic attack of GSH on caftaric acid O- quinones which is induced by polyphenol oxidase in the presence of oxygen immediately after crushing NH SO3 COOH 2 H O O N grapes HOOC N COOH HO COOH H O O OH S O COOH HO • A large portion of free sulfur dioxide is hydrated at wine pH and therefore present in the form of the HO E COOH O reaction products? bisulfite ion (HSO ˉ) which is effective against oxidation OH 3 HO 2-S-glutathionyl-(E)-caftaric acid • Therefore, reaction products of bisulfite and caftaric acid in the O-quinone form are likely present in (Grape Reaction Product, GRP) wine HPLC chromatograms of a commercial wine monitoring [M-H]¯ To discover the reaction products using high resolution mass spectrometry (HPLC at m/z 390.998± 0.005 and UV absorption at 320 nm QTOF-MS analysis) : m/z 390.998 ± 0.005 : 320 nm absorbance a The following assumptions were made: 1. Chemical formula C13H12O12S as reaction products of caftaric acid (C13H12O9) and bisulfite (H2SO3) b 2. 320 nm absorbing compounds as caftaric acid derivatives 3. Sulfonic acid derivatives are strong acidic compounds c Five compounds, a-e were found in a commercial wine Intensity e d The compounds, a-e exhibit the same expected [M-H]¯ ion (m/z 390.998, C13H11O12S), 320 nm absorption and acidic property as confirmed by an anion exchange resin, therefore they are likely the reaction 4 6 8 10 12 Time [min] products. Product ion (MS/MS) spectra of the compounds a-e To confirm the compounds a-e as the reaction products based on their fragment ions Intens. 4 x10 4 240.981 390.997 a m/z 179.035 [C H O ] m/z 258.992 [C H O S] 149.009 9 7 4 ‒SO 9 7 7 133.030 161.025 212.986 258.992 3 ‒CO2 [M-H]¯: m/z 390.998 [C H O S] x104 HO 13 11 12 4 390.998 b m/z 135.045 [C8H7O2] 161.025 240.981 OH ‒SO 149.009 3 179.034 212.981 258.991 O OH m/z 133.030 [C8H5O2] HO x104 m/z 311.040 [C H O ] 3 161.025 390.997 O O 13 11 9 c ‒SO3 240.980 SO3H O OH 215.001 ‒CO 149.010 258.991 m/z 212.986 [C8H5O5S] 5000 149.009 m/z 161.0244 [C9H5O3] m/z 240.981 [C9H5O6S] m/z 149.010 [C4H5O6] 390.998 d ‒SO3 179.036 258.988 311.045 Major fragment ions of a-e indicate the involvement of caftaric acid, caffeic acid, tartaric acid and/or 390.996 4000 149.009 e bisulfite in their structures, supporting that these compounds are the reaction products of caftaric acid 179.036 240.978 258.989 311.040 and bisulfite. 150 200 250 300 350 400 m/z Structural identification of the reaction compounds a-e How much are the reaction compounds a-c found in commercial wines? with the aid of NMR and UV spectroscopic techniques Chardonnay (n=10, Vintages: 2012-2015) 1' (mg/L)* Reaction products a-c Grape reaction product Caftaric acid O COOH OH 2 7 E Average 29 28 39 HO 3 3' COOH O S O 9 2' 1 8 O 4' 2 Median 30 28 37 HO Z 6 OH COOH HO 4 Max 43 43 73 5 COOH O S O HO O O Min 11 6 6 OH OH a: 5-sulfo-(E )-caftaric acid b: 2-sulfo-(Z)-caftaric acid Cabernet Sauvignon (n=11, Vintages: 2012-2015) (mg/L) Reaction products a-c Grape reaction product Caftaric acid OH O COOH Average 29 16 83 O S O O COOH HO E COOH 2 O Median 29 16 75 HO E COOH O O OH HO S O 4 Max 46 21 141 OH HO O Min 15 9 46 c: 2-sulfo-(E)-caftaric acid d: (E)-caftaric acid-4-O-sulfate * mg/L (caftaric acid equivalent) O O COOH E HO S O 3 COOH The reaction products in commercial white and red wines are found to be present as O O much as GRP, suggesting that bisulfite consistently competes with glutathione for OH HO nucleophilic caftaric acid. e: (E)-caftaric acid-3-O-sulfate The AWRI is a member of the Wine Innovation Cluster.