Optimization and validation of an alternative method to evaluate total reactive antioxidant potential.
The total reactive antioxidant potential (TRAP) is one of the methods most employed to estimate the antioxidant capacity of samples in vitro. This method is based on the quenching of luminol-enhanced chemiluminescence derived from the thermolysis of 2,2'-azo-bis(2-amidinopropane)dihydrochloride (AAPH) as the free radical source. However, this method can present limitations when the sample does not present a lag phase. In addition, there are no studies regarding TRAP assay validation. In this context, the aim of this work was to optimize and validate this method and to propose another evaluation method using the area under the curve (AUC). The main condition established was the need for the stabilization of the system, at 7000s, before the addition of the antioxidant to be tested. Both evaluation methods were validated using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as a calibrator in the range of 50 to 250nM, and all parameters showed satisfactory results: specificity, linearity (r>0.99), precision (intra and interassay relative standard deviations <5%), robustness, and the limits of detection and quantitation (low and similar for both methods). The main advantage of the use of AUC is to evaluate the antioxidant potential of samples that do not present lag phase.
Dresch
Maria T K
MT
Programa de Pós-Graduação em Ciências Farmacêuticas, Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), 2752 Porto Alegre, Brazil.
Rossato
Simone B
SB
Kappel
Vírginia D
VD
Biegelmeyer
Renata
R
Hoff
Mariana L M
ML
Mayorga
Paulo
P
Zuanazzi
José A S
JA
Henriques
Amélia T
AT
Moreira
José C F
JC
D000578:Amidines / Q000737:chemistry; D000975:Antioxidants / Q000032:analysis*; D019540:Area Under Curve; D002138:Calibration; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002839:Chromans / Q000737:chemistry; D005609:Free Radicals / Q000737:chemistry; D016014:Linear Models; D008163:Luminescent Measurements / Q000379:methods*; D008165:Luminol / Q000737:chemistry; D012680:Sensitivity and Specificity; D013816:Thermodynamics; D013997:Time Factors
D000578:Amidines; D000975:Antioxidants; D002839:Chromans; D005609:Free Radicals; D008165:Luminol; C046728:2,2'-azobis(2-amidinopropane); C010643:6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid