Identification of reaction products of 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 are strong nucleophilic compounds (electron donors) and act as antioxidants in HOOC N COOH H bisulfite ion O wine HS Glutathione (GSH) • (GRP) can be formed by a nucleophilic attack of GSH on caftaric acid O- quinones which is induced by 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, , 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

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