*3.3. Vitamin C and Urate Analysis*

Plasma, media, and FBS samples were prepared for ascorbate analysis using HPLC as previously described [134]. Briefly, acid extracts were diluted in sodium acetate/methanol/water mobile phase (0.3%/7.5%/92% w/v) containing Q12 ion pairing reagent and pH-adjusted with 2.58 M KH2PO4 buffer (pH 9.8). Samples were separated on Watters 2695 using an LC-8 column (Supelco) under an applied potential of 600 mV using an electrochemical detector (BSA). Under these conditions, both urate and ascorbate are resolved and quantified by comparisons to authentic standards with a detection limit of approximately 1 nM. For human plasma samples, urate values are used to normalize ascorbate values to minimize any variations within subject samples and to control for volume differences in vacutainer analyses. To obtain total ascorbate, samples were reduced with the addition of tris-2-carboxyethyl phosphine (TCEP) as described elsewhere [113]. Dehydroascorbic acid content is estimated by the difference between TCEP-reduced and standard preparations.
