Antioxidant Activity Test

Method description

The antioxidant activity of foods is determined using a method designed to give maximum extraction of the antioxidant capacity of the sample. The stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical gives a strong absorption maximum at 517 nm and is purple in color. The color turns from purple to yellow when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant forming the reduced DPPH-H. The resulting decolorization is stoichiometric with respect to the number of electrons captured. The reference standard, Trolox [(S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid], and the sample are extracted and reacted with the methanol-water DPPH solution for four hours at 35°C and the absorbance changes are measured at 517 nm. The quantity of sample necessary to react with one half of the DPPH is expressed in terms of the relative amount of Trolox reacted. Antioxidant activity of a sample is expressed in terms of micromole equivalents of Trolox (TE) per 100 grams of sample, i.e. Trolox units per 100 g or TE/100g.

Acceptable matrices

This method is applicable to solid and liquid samples.

Unacceptable matrices

Fats and Oils

Limit of quantitation

400-400,000 micromoles of Trolox equivalents (TE) per 100 grams of sample

Equipment

Spectrophotometer

Method reference

AOAC 2012.04 – “Estimation of Antioxidant Activity in Foods and Beverages by Reaction with 2,2′-diphenyl-1-picrylhydrazyl (DPPH)”

Reportable unit

TE/100g

Sample size requirements

50 grams

Information required by submitter

Please supply estimates.

Additional information

Antioxidant activity of foods is measured by simultaneously extracting the food’s antioxidants and reacting them with the stable radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Since a separate extraction step is not used, the simultaneous extraction/reaction step facilitates the extraction process to completion and also allows measurements to be taken before degradation of the extracted material. In this method, DPPH is allowed to react with the whole sample and sufficient time allows reaction with weak antioxidants. Results are expressed as Trolox Equivalents by using the stable antioxidant Trolox as a calibrating agent. Other methods, such as ORAC, measure total antioxidant activity by first extracting the food’s antioxidants into 1 or more solvents before reaction with radicals. The DPPH and ORAC values will differ but trending within a food set is possible by both.