Insights on Antioxidant Assays for Biological Samples Based on the Reduction of Copper Complexes—The Importance of Analytical Conditions
High-throughput cupric ion reducing antioxidant capacity (CUPRAC) methods were developed for assessment of total antioxidant capacity (TAC) in urine and serum, based on reduction of Cu(II)-neocuproine complex to highly colored Cu(I)-neocuproine complex, measured spectrophotometrically at 450 nm. The reaction time was significantly reduced from 30 to 4 min by application of a calibration compound (uric acid) with kinetic behavior similar to that shown by urine samples. The method was implemented in a microformat (96 well plates) and also in an automatic fashion (flow injection analysis, FIA). A determination throughput value of 288 h(-1) (microplate method) or of 15 h(-1) (automatic FIA) was attained. Application of both methods to human serum (SRM 909b, level I) and urines (n = 9) provided TAC values in agreement with those of the end-point batch method.