4.4. Sample Preparation and Analysis

Wine samples and all stock solutions were prepared in a low oxygen atmosphere by making use of an anaerobic bag (Aldrich^® AtmosBag, Sigma-Aldrich, Castle Hill, NSW, Australia) fitted with a PreSens Pst6 oxygen sensor (Presens, Regensburg, Germany). The bag was flushed down to <1 ppb oxygen with N_{2 (g)} at the start of the sample preparation and a slight positive pressure was maintained to prevent oxygen ingress into the bag during experimental setup. The oxygen concentration in the anaerobic bag was continuously monitored and maintained at <15 ppb. The Verdelho, Shiraz, and model (12% EtOH, 10 g/L tartaric acid, pH = 3.4) base wines were placed inside the anaerobic bag and sub-sampled by placing 10 mL of wine into a 20 mL crimp top amber vial (Chromacol, Thermo Fisher Scientific Inc., Scoresby, VIC, Australia). The Verdelho and Shiraz wines were each divided into four treatments repeated in triplicate (n = 12): (1) Cu²⁺ treatment (1 mg/L for Verdelho, Shiraz and model wines); (2) SO₂ treatment (Verdelho: free 87.8 mg/L, total 100.0 mg/L; Shiraz: free 40.8 mg/L, total 60 mg/L; model wine: free 99.67 mg/L, total 100 mg/L); (3) “Cu²⁺ + SO₂” treatment, where the above mentioned levels of Cu²⁺ and SO₂ were combined into one treatment for Verdelho, Shiraz, and model wines; and (4) the control Verdelho, Shiraz, and model wine samples were not treated with either Cu²⁺ or SO₂ (Table 3).

Stock solutions of Cu²⁺, and SO₂ were prepared in such a manner that the high level of each metal was added by spiking 50 µL of the appropriate stock solution to the 10 mL wine sample using a pipette, avoiding significant dilution effects. After the samples were prepared with the four treatments, they were sealed with 20 mm magnetic crimp caps with 8 mm center and blue PTFE/silicon septa (118 mm) (Grace Davison Discovery Sciences, Rowville, VIC, Australia) and stored at room temperature (20 °C) in 19 L post mix Cornelius kegs (Ambar technology, Alexandria, NSW, Australia). The lid of each keg was fitted with a PreSens Pst6 oxygen sensor (Presens, Regensburg, Germany). The kegs were flushed with N_{2 (g)} until they reached <1 ppb oxygen and maintained at a slight positive pressure of 1 psi N_{2 (g)} to prevent any oxygen ingress into the keg storage systems. The kegs were continuously monitored and not allowed to exceed an oxygen measurement of 25 ppb, and flushed with N_{2 (g)} to <1 ppb if increased oxygen concentrations were measured.

Verdelho, Shiraz, and model wine samples were analyzed for their VSC concentrations immediately after spiking with metals (Day 0), and then following 1 month (Month 1), 3 months (Month 3), 6 months (Month 6) and 12 months (Month 12) of storage under low oxygen conditions.