High-throughput Total Cupric Ion Reducing Antioxidant Capacity of Biological Samples Determined Using Flow Injection Analysis and Microplate-based Methods

Abstract

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.

High-sensitivity programmable flow method for assessment of total antioxidant capacity in biological samples

“…Recently, in mesofluidic, miniaturised, automated systems using minimum sample volume (1 μL), an oxidant to oxidant ratio of 126 times could be , as reported for neocuproine complexes [16,30].…”

“…The stock solution of 1.00 mM uric acid (UA) was prepared by suspending the corresponding solid in 20 mL water, the pH of the resulting solution was raised by dropwise addition of 0.1 M NaOH solution until complete dissolution of the solid (pH [11][12], followed by the elimination of the excess of base by addition of 0.1 M HCl solution until reaching pH 7.0. Afterwards, the total volume of the solution was completed to 100 mL with water [16]. Working standard solutions of antioxidants in the range of 1 to 10 μM were prepared by adequate dilution in water.…”

“…Considering that, in general, for batch total antioxidant capacity assays [16] the oxidant to antioxidant ratio lies between 10 and 100 times (depending on the antioxidant concentration), the use of higher oxidant excess was up to now impracticable due to limitations in detection limits (as low antioxidant concentrations should be targeted). Recently, in mesofluidic, miniaturised, automated systems using minimum sample volume (1 μL), an oxidant to oxidant ratio of 126 times could be , as reported for neocuproine complexes [16,30].…”

“…TAC assays have been adapted to microplate format aiming to attain high-throughput results suitable for bioanalysis purposes [5,16,17]. TAC assays are extremely affected by reaction conditions, fostering noncomparable results between studies [18].…”

“…Automation of TAC assay is beneficial in providing strict control of reaction conditions, including reaction time. The literature describes strategies that include microplate and flow injection assays with matrix matching approach [16] and flow through spectrophotometric method with kinetic assessment [19]. The general downscaling of the assay also provides reduced sample consumption, as illustrated by the semi-micro FIA microdialysis probe hyphenated system that was applied for evaluation of the ex vivo antioxidant effect of the microdialysate after ascorbic acid administration in rats for preclinical assays [20].…”