Concentration and purification of grape extracts

When the thiol precursors (usually only G4MMP) were below analytical limits for evaluation by direct injection, grape extracts were concentrated and purified to eliminate tannins and sugars which would impact the integrity of mass spectrometry. Extraction was performed using a Dowex 50WX4–100 ion exchange resin in a micro-column, with a modified method as previously described for grape juice and must²⁵. Methanol was evaporated from the extracts under reduced pressure at 38 °C, the residue was diluted in water (4 mL) and quantitatively loaded onto 160 mg of ion exchange resin, previously conditioned with water (1.2 mL), followed by 2 M HCl (1.2 mL) and water (6 mL). The sample was washed with water (6 mL) and eluted with 4 mL of 1 M ammonium buffer (NH₄H₂PO₄). The eluent was subsequently loaded onto a hydrophobic cartridge (6 mL, 500 mg, Strata SDB-L), previously conditioned with methanol (6 mL) and 5 mM H₂SO₄ (6 mL). The cartridge was washed with 5 mM H₂SO₄ (5 mL) and dried under a flow of nitrogen. Thiol precursors were eluted with methanol (5 mL). Methanol was evaporated to dryness under reduced pressure at 38 °C and the residue was immediately redissolved in 200 µL mixture of methanol:water 1:1, filtered through a 0.22 μm PVDF filter and analysed using UHPLC-MS/MS.

To assess recovery of thiol precursors during the purification process, grape extracts (n = 6) were spiked with a thiol precursor mixture (5 µL, 25 mg L⁻¹ in 50% aqueous methanol), vortexed and centrifuged. A small aliquot was filtered and directly injected into UHPLC-MS/MS. Grape extracts were then purified according to the procedure (see above) and recovery of purification was determined with regard to direct injection. The average recoveries (±standard error of the mean, n = 6) of Cys4MMP-d ₆, Cys4MMP, Cys3MH, G4MMP-d ₁₀, G4MMP, and G3MH from grape extracts during purification were 69.0 ± 2.3%, 66.2 ± 3.0%, 76.8 ± 8.9%, 76.4 ± 8.1%, 71.4 ± 8.7% and 73.3 ± 6.9% respectively.