47.2.

06
AOAC Officiaf Method 2012.04
Antioxidant Activity in Foods and Beverages
Reaction with 2,2'-Diphenyl-1-Picrylhydrazyl (DPPH)
First Action 2012
[Applicable to tl1e deter1ni11ation of nntioxiclants in toocls a11d
beverages by reaction witl1 2,2'-cfiphenyl-1-JJicrylhyclr,tzyl ( DPPJ-1). J

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 See Tables 2012.04A and 8 for rcsults of the interlaboratory study
supporting acceptance of thc n1cthod.
A. Principie

The ,nethod detern1ines the antioxidant acttv1ty of foods and

beverages by reaction ,vith the stablc radical DPPH. This method is
not applicable to high-fat a11d -oil rnatrixcs (>50% fat). The DPPH
free radical gives a strong absorptio11 1naxi111un1 at 517 11111 and is
purple in color. The color tun1s frorn purple to yellow as the n1olar
absorptivity of the DPPH radical at 517 11111 decreascs from 9660 to
1640 µM/c111 ,vhcn the odd electron of DPPH radical becomes paired
with an electron donated fron, an antioxida11t and a hydrogen atom
(hydride equivalent) to fonn the reduced DPPH-H. The resulting
decolorization is stoichion1etric ,vith respect to nt11nber of hydride
equivalents captured.
Antioxidant cornpounds 111ay be \vater-soluble, lipid-soluble,
insoluble. or bound to cell ,valls. Thus, extraction efficiency is an
in1portant factor in the quantification of antioxidant activity of foods.
This n1ethod n,aximizes the extraction efficiency by adding the
DPPH standard solution directly to the sample. A separate extraction
step is not perfo1111ed. Antioxidant co1npounds are continually
extracted fron1 the sa,nple and iinn,ediately reacted with DPPH until
the extraction and reaction are con1plete. The sample is then filtered,
and the absorbance change is detennined. Calibration is achieved
as described below by co,nparison to solutions of kno\vn Trolox
concentration.
The san,ple and Trolox, a vitamin E analog which is used as the
reference standard, are reacted ,:vith DPPH solution in rnethanol­
water for 4 h at 35 ºC in a vessel mounted on a rotary shaker. and
the absorbance changes are n1easured al 517 nn1. The quantity of
sa111ple necessary to react with one-half of the DPPH is expressed in
terms of the r�lative an,oun� of rrrolox reacted. Antioxidant activity
of a sa,nple 1s expressed 1n terms of �Lmole Trolox equivalents
(TE)/100 g san,ple.
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