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

## TL;DR

A new system combines two antioxidant tests into one efficient method for measuring antioxidant capacity in food and biomedical samples.

## Contribution

A novel sequential injection analysis system integrates FRAP and ABTS assays for simultaneous antioxidant evaluation with improved efficiency and reagent savings.

## Key findings

- The FRAP/ABTS-SIA system reduced reagent use by 70% for FRAP and 50% for ABTS compared to traditional methods.
- The method achieved high precision (RSD < 2%) and strong correlations (≥0.99) with reference assays in complex food samples.
- It enables a throughput of ∼30 samples per hour with detection limits as low as 0.0047 μmol L–1 for ABTS.

## Abstract

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.