A Breakthrough SIA-Based Dual Assay for Simultaneous Evaluation of Antioxidant Capacity via ABTS and FRAP Mechanisms

An innovative method, termed FRAP/ABTS-SIA, was developed to simultaneously integrate the FRAP and ABTS antioxidant assays within a single sequential injection analysis (SIA) system with spectrophotometric detection. Leveraging the kinetic differences between the assays and controlling the dispersion, a compact aspiration sequence (antioxidant-FRAP-ABTS-antioxidant-water) was optimized using a central composite design, defining a flow rate of 40 μL s^-1 and aspiration volumes of 43, 38, 38, 43, and 100 μL, respectively. The system incorporated a helical reaction coil positioned before the detector, allowing the antioxidant-FRAP bolus to react while the ABTS-antioxidant-water sequence was aspirated into the holding coil. This configuration enhanced the FRAP signal and enabled clear separation of both analytical responses. Compared to conventional batch protocols, this strategy reduced FRAP reagent concentrations by 70% and ABTS^•+ radical concentrations by 50%. The method delivers responses within a 2 min run, achieving a throughput of ∼30 samples h^-1. Linearity was confirmed for both assays over the range 10-120 μmol L^-1 Trolox, with detection limits of 0.031 μmol L^-1 (FRAP) and 0.0047 μmol L^-1 (ABTS). Intralaboratory precision was below 2% RSD, and recoveries ranged from 97.3 to 106.2% (FRAP) and 92.8 to 105.4% (ABTS). The method was successfully applied to complex food matrices─including coffees, wines, juices, and spices─showing correlations ≥0.99 with microplate reference assays. High-throughput, reagent savings, metrological robustness, and simplified data processing position FRAP/ABTS-SIA as an efficient and reliable tool for routine antioxidant capacity evaluation in food and biomedical applications.