Measurement of Total Antioxidant Capacity of human plasma: setting and validation of the CUPRAC-BCS method on routine apparatus ADVIA 2400

Background: Quantification of Total Antioxidant Capacity (TAC) of human plasma is an important clinical target, since many diseases are suspected to be related with oxidative stress. The CUPRAC-BCS (BCS = Bathocuproinedisulfonic acid) method was chosen since it works using the photometric principle, with stable and inexpensive reagents and at physiological pH. Methods: The method is based on the complex equilibria between Cu(II)-BCS (reagent) and Cu(I) -BCS. Cu(I)-BCS complex is formed by reducing ability of the plasma redox active substances. The photometric signal is achieved at 478 nm and calibration is performed using urate as reference substance. Results: Linearity, linear working range, sensitivity, precision, LoD, LoQ, selectivity and robustness have been considered to validate the method. Absorbance at 478 nm was found linear from 0.0025 up to 2.0 mmol L-1 of urate reference solution. Precision was evaluated as within-day repeatability, Sr = 4 µmol L-1, and intermediate-precision, SI(T) = 15 µmol L-1. LoD and LoQ, resulted equal to 7.0 µmol L-1 and 21 µmol L-1 respectively while robustness was tested having care for pH variation during PBS buffer preparation. Tests on plasma (80 samples) and on human cerebrospinal fluid (30 samples) were conducted and discussed. Conclusions: By the analytical point of view, the photometric method was found to be simple, rapid, widely linear and reliable for the routine analysis of a clinical laboratory. By the clinical point of view, the method response is suitable for the study of chemical plasma quantities related to redox reactivity.