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Method for the Determination of Antioxidant Activity in Foods and Beverages

by Reaction with 2, 2’-diphenyl-1-picrylhydrazyl (DPPH): Collaborative
Study
David W. Plank
Medallion Labs, 9000 Plymouth Avenue North, Minneapolis, MN 55427

John Szpylka
General Mills, Inc., 9000 Plymouth Avenue North, Minneapolis, MN 55427

Harry Sapirstein
University of Manitoba, Dept of Food Science and Nutrition, Winnipeg, Manitoba, MB R3T 2N2, Canada

David Woollard
NZ Laboratory Services Ltd, 35 O'Rorke Road, Penrose, Auckland 1642, New Zealand

Charles M. Zapf
McCormick & Company, Inc., Technical Innovation Center, 202 Wight Avenue, Hunt Valley, MD
21031

Vong Lee
POM Wonderful, LLC, 5286 S. Del Rey Ave, Del Rey, CA 93616

Oliver Chen
Tufts University, Antioxidant Research Lab, 711 Washington St., Boston, MA 02111

Rui Hai Lui

Cornell University, Dept. of Food Science, 108 Stocking Hall, Cornell University, Ithaca, NY 14853

Rong Tsao
Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Road West, Guelph,
Ontario N1G 5C9

André Düsterloh
DSM Nutritional Products, Ltd, Bldg 205, Rm 6, Wurmisweg 576, 4303 Kaiseraugst / Switzerland
________________________

Corresponding author’s email: david.plank@medlabs.com

________________________

Collaborators: A. Begelman; M. Camire; M.P. Dougherty; C. DeRito; M. Hanson; M. Marquardt; J.W.

DeVries; Ronghua Liu

Abstract/Summary

A collaborative study was conducted to evaluate a colorimetric method for the determination of total
antioxidant activity in a variety of foods, beverages and oils. The procedure involved the extraction of the
antioxidants directly into a methanol/water solution containing a known amount of 2, 2’-diphenyl-1-
picrylhydrazyl (DPPH), thus promoting the rapid reaction of extracted materials with DPPH. The reaction

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was monitored by spectrophotometric measurement of the absorbance loss at 517 nm. Antioxidant activity
was quantified relative to a dilution series of Vitamin E analogue standards (Trolox®) which were analyzed
in parallel simultaneously with the food, beverage and oil samples.

Nine laboratories analyzed twenty-two (22) samples comprised of eleven blind duplicates for Total
Antioxidant Activity. The antioxidant activities of the samples ranged from 131 and 131,000 µmol Trolox
Equivalents (TE)/100g. Statistical analysis of the results showed that nine of the eleven matrices gave
acceptable HORRAT values indicating that the method performed well in these cases. The acceptable
matrices include pomegranate juice, blueberry juice, carrot juice, green tea, wine, rosemary spice, ready-to-
eat cereal, and yogurt. There were two samples that failed the HORRAT test, the first being an almond
milk that had an antioxidant level below the practical limit of quantification for the method. The second
was a sample of canola oil with added omega-3 fatty acid that was immiscible in the reaction medium.
Based on the results of this study, the method is recommended for adoption as Official First Action.

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Introduction

Antioxidant compounds in food play an important role as a health-protecting factor. Studies suggest
that antioxidants reduce the risk for chronic diseases including cancer and heart disease (1). Primary
sources of naturally occurring antioxidants are whole grains, fruits, and vegetables. Plant sourced food
antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytate and phytoestrogens have been
recognized as having the potential to reduce disease risk (2-5).

The main characteristic of an antioxidant is its ability to trap free radicals. Highly reactive free radicals
and oxygen species are present in biological systems from a wide variety of sources. These free
radicals may oxidize nucleic acids, proteins, lipids or DNA and can initiate degenerative disease.
Antioxidant compounds like phenolic acids, polyphenols and flavonoids scavenge free radicals such as
peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to
degenerative diseases.

It is impossible or impractical to measure each antioxidant in foods or biological samples so generic

tests are required that collectively determine all the active materials without individual identification.
This leads to a number of different methods based upon changes in chemiluminescence, electron spin
resonance (ESR) or spectrophotometry. Early methods often would require special equipment and
technical skills for the analysis. These analytical methods measure the radical-scavenging activity of
antioxidants against free radicals like the 1,1-diphenyl-2-picrylhydrazyl radical, the superoxide anion
radical, the hydroxyl radical, or the peroxyl radical. There are other methods that determine the
resistance of lipid or lipid emulsions to oxidation in the presence of the antioxidant being tested. The
malondialdehyde (MDA) or thiobarbituric acid-reactive-substances (TBARS) (7) assays have been used
extensively since the 1950’s to estimate the peroxidation of lipids in membrane and biological systems.
These methods can be time consuming because they depend on the oxidation of a substrate which is
influenced by temperature, pressure, matrix etc. and may not be practical when large numbers of
samples are involved.

Antioxidant activity methods using free radical traps are relatively straightforward to perform. The
ABTS [2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)] radical cation (8) has been used to screen
the relative radical-scavenging abilities of flavonoids and phenolics through their properties as electron
or proton plus electron donating agents. Prior et al. (9) have used the Oxygen Radical Absorbance
Capacity (ORAC) procedure to determine antioxidant capacities of fruits and vegetables. In the ORAC
method, a sample is added to the peroxyl radical generator, 2,2’-azobis(2-
amidinopropane)dihydrochloride (AAPH) and inhibition of the free radical action is measured (10)
using the fluorescent compound, B-phycoerythrin, R-phycoerythrin or fluorescein. Vinson et al. (11)
have measured phenolics, a significant class of plant-based antioxidants, in fruits and vegetables using
the Folin-Ciocalteu colorimetric reagent using inhibition of low density lipoprotein oxidation mediated
by cupric ions.

The use of the free radical, 2,2-Diphenyl-1-picrylhydrazyl (DPPH) is widely used to test the ability of
compounds to act as free radical scavengers or proton plus electron donors, and to evaluate antioxidant
activity of foods and other complex biological systems. Comparison with the Vitamin E analogue,
Trolox (S)-(-)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is the usual way to express
results (i.e. µmolTE/100g). In this study the complete sample is continuously reacted with DPPH in
methanol/water for 4 hours at 35 C facilitating full extraction and reaction of most antioxidant
compounds, including fat-soluble materials. The method is applicable to solid or liquid samples and is
not specific to any particular antioxidant component, but applies to the overall antioxidant capacity of
the sample. The reaction kinetics for specific antioxidants have been published and the stoichiometry
characterized (12,13). DPPH is reduced by the antioxidants through the transfer of a proton plus an

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electron (a hydride equivalent) resulting in color-loss of the DPPH. To appreciate the antioxidant
property of any sample, the color-loss cannot always be taken to completion therefore the weight of
sample is adjusted so that approximately 50% of DPPH color is lost. This requires a pre-knowledge of
the sample or alternatively preliminary tests to decide the most appropriate sample weight.

Determination of antioxidant activity of foods using DPPH is qualitatively comparable to other methods
but can differ in absolute data even when each involves utilization of Trolox for calibration. It is
probable each of these methods measure a somewhat different profile of antioxidant compounds
depending on the nature of the food, the selected solvent and the measurement process. However, when
one studies identifiable compounds with similar structures, these compounds follow similar kinetic
trends no matter which of the various methods is used (14-18).

There are many nutritional claims made by food manufacturers concerning the antioxidant status of
their foods. A method is required to substantiate these claims, preferably one that is accepted globally
so comparisons between labels are valid. This study has been undertaken to establish the accuracy,
between lab reproducibility and utility of the DPPH antioxidant method as a reliable, standardized
method for assessing the antioxidant capacity of foods and beverages.

Summary of Single Laboratory Validation Study

In advance of the collaborative study, a single laboratory validation was performed to assess the
method’s accuracy, precision, and instrument response range.

Method Accuracy – The accuracy of the method was assessed by spiking three representative matrices
with ascorbic acid. Cocoa powder and pomegranate juice represented the spiking of matrices with relevant
antioxidant activities. Corn starch represented the spiking of a low-level control.

The pomegranate juice was spiked directly with ascorbic acid at four levels representing approximately
25%, 50%, 75%, and 100% of the indigenous amount. To facilitate the spiking process of the cocoa
powder and corn starch flour, a 1.7% ascorbic acid in corn starch powder blend was used as the spiking
agent. This blend was kept frozen until used where it was warmed to room temperature prior to spiking the
food matrices.

An overall recovery for each matrix was calculated using the slope of the least-squares best-fit linear
regression from a plot of the analytical result (y-axis) against the equivalent spike amount (x-axis). The
results of this accuracy study are summarized in Table 1. This data demonstrates the method is
sufficiently accurate.

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a
Table 1.  Results of single lab method accuracy study: recovery of ascorbic acid

Number of  Antioxidant Activity of Spikes Coefficient of 

Sample Recovery, % 2
Spikes, n (μmol TE/100g) determination, R

b
Corn Starch 5 485; 5840; 11886; 18734; and 24894 114.60 0.9941
Cocoa Powder 5 126602; 149878; 167042; 219286; and 224627 92.63 0.9461
Pomegranate 5 3488; 4158; 5150; 5861; and 6934 106.40 0.9933

a
 Antioxidant activity of ascorbic acid determined to be 582,211 μmol TE/100g
b
 Activity range includes unspiked sample

Method Precision – The precision of the method was assessed on ten matrices. Each matrix was analyzed
in triplicate on each of three separate days, thereby obtaining nine Antioxidant Activity results for each
matrix. This was performed by two technicians each analyzing all ten matrices at separate times. The
precision study is summarized in Table 2 and adequately demonstrates the reliable repeatability of this
method for a variety of food matrices.

Table 2.  Results of single lab method precision study (μmol TE/100g reported by laboratory)

Blueberry  Pomegranate  Pomegranate  Pomegranate  Pomegranate 

Day / Replicate Cocoa RTE cereal Green tea Carrot juice Red wine
juice juice concentrate by‐product arils

1 / 1 119,392 3,107 1,795 822 379 3,093 3,492 9,938 199,985 16,188
a
1 / 2 117,390 3,147 1,938 843 394 3,031 3,512 9,826 256,657 17,713
1 / 3 112,408 3,234 1,943 826 390 2,842 3,525 10,212 204,535 17,090
2 / 1 120,003 3,049 1,917 803 375 2,961 3,664 10,101 216,800 19,145
2 / 2 121,937 3,087 1,921 810 372 3,268 3,617 10,075 208,899 17,327
2 / 3 114,366 3,038 1,985 803 350 3,089 3,703 9,910 204,339 17,821
3 / 1 127,784 3,093 1,938 845 371 2,943 3,449 9,718 197,172 18,680
3 / 2 127,446 3,071 1,893 849 378 3,074 3,071 9,750 185,353 17,278
3 / 3 119,581 3,085 1,927 840 381 3,162 3,386 9,854 188,794 18,184

Average 120,034 3,101 1,917 827 377 3,051 3,491 9,932 200,735 17,714
Sr 5202 59 52 18 13 126 188 167 10314 885
RSDr , % 4.33 1.91 2.72 2.22 3.35 4.13 5.37 1.69 5.14 5.00
b
HORRAT Value 0.79 0.20 0.27 0.19 0.26 0.43 0.57 0.21 1.01 0.68
a
 Statistical outlier.
b
 HORRAT value was evaluated based on the finite mass of Hydride ions equivalent to the moles of Trolox oxidized per mole DPPH reduced (i.e. 10‐8 g hydride ions/g sample is 
equivalent to 1 μmole TE/100g sample).

Instrument Response Range – A Trolox® standard solution (50.00 mg in 100 ml of 50% methanol/water)
was assayed four times at each of multiple Trolox® amounts: 0, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 mg.
The best-fit line for the average values plotted versus mg Trolox® had an R2 value of 0.9925. The
portion of the curve, from 0.1 to 0.5 mg (0.1 to 0.75 absorbance units) has an R2 value of 0.9998. This
demonstrates that the instrument response of the levels of Trolox® equivalents typically observed is
adequately linear for quantification.

Calculation of method limit of quantification (LOQ) – Calculation of the method LOQ was performed
using the two equations described in the method calculations. Combining both equations yields:
Antioxidant Activity = [(391546) x (Absorbance Target)] / [(Target Mass) x (Trolox Slope)]. Using the
Trolox calibration curve, an average “Absorbance Target” is ½ of an average y-intercept of 0.911
absorbance units and the average “Trolox Slope” is 1.5415. Thus, LOQ = [1 g Trolox/1000 mg Trolox]
x [(391546 µmol TE/100g Trolox) x (1/2 x 0.911 abs)] / [(0.5 g sample) x (1.5415 abs/mg Trolox)] =
2.31 µmol TE / g sample or 231 µmol TE / 100 g sample.

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Collaborative study

Precollaborative Ruggedness Study

A precollaborative ruggedness study was conducted with the participating laboratories to ensure adequate
method performance. Labs participating in the evaluation were sent three samples packed under an Argon
layer and sealed by septa along with copies of the method, and 1 gram each of DPPH [2,2’-Diphenyl-1-
(2,4,6-trinitrophenyl) hydrazyl] and Trolox [6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid].
Each laboratory was requested to run each sample in singlet. Laboratories were requested to conduct the
analysis, ask questions regarding procedures and provide feedback to the Study Directors on any aspects of
the method about which the collaborator might have a concern. The results of the analyses on the
ruggedness testing samples are shown in Table 3. Relevant comments received from the participating
laboratories were incorporated as changes to improve the method as appropriate. No significant procedural
changes were found to be necessary.

Table 3.  Results of precollaborative ruggedness testing (μmol TE/100g reported by laboratories)
Sample / Sample No.
Pomegranate  Pomegranate  Oat Cereal 
Juice Juice Base
Lab Number K31610 P31510 RM25F

a
1 3533 4368 2785
2 3768 4314 2608
3 3566 3673 1777
4 3312 3398 1570
5 3504 3610 2181
6 3201 3264 1659
7 3900 4000 2000
8 3624 3683 2353
9 3564 3776 NR

Average 3552 3787 2021

SR 211 378 382
RSDR, % 5.94 9.97 18.89
b
HORRAT Value 0.64 1.08 1.86

NR = Not Reported.
a
 Statistical outlier.
b
 HORRAT value was evaluated based on the finite mass of 
Hydride ions equivalent to the moles of Trolox reduced 
‐8
(i.e. 10  g hydride ions/g sample is equivalent to 1 µmol 
TE/100g sample).

The results for the precollaborative ruggedness study gave a HORRAT value for all samples greater than
0.5 but less than 2 which indicates that the laboratories’ analysis results meet the criteria for acceptable
method performance in accordance with AOAC guidance (19-21). Based on the acceptable results of
precollaborative ruggedness study, the Study Directors determined that the method was ready for a full
collaborative study.

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Collaborative Study Protocol

Eleven food samples representing products commonly marketed for their antioxidant activities were
selected for the collaborative study for analysis as blind duplicates by the participating laboratories. The
matrices ranged from a variety of beverages (almond milk, blueberry Juice, carrot juice, green tea,
pomegranate juice and red wine) to fermented dairy (yogurt) to ground solids (rosemary and ready-to-eat
(RTE) cereal) to oils (canola oil plus omega-3).

All samples were coded and randomized to ensure the blind nature of the duplicates for each matrix as
shipped by the Study Directors. The samples were all prepared to a homogenous distribution prior to sub-
sampling. The samples were packed in individual 40 mL glass vials and sealed with septa. An argon flush
in the head space above each sample was performed and an argon blanket was left over each sample.
Samples were shipped to each laboratory by overnight air with instructions to transfer samples to 4C
storage conditions in the dark on arrival.

A total of nine laboratories reported data for the collaborative study samples. An additional laboratory had
to withdraw due to samples arriving while the principle investigator was not available. Samples sat at room
temperature for a period of 3 weeks prior to analysis. Clear indications of aberrant results for the samples
led the lab to request withdraw from the study. Data from the withdrawn laboratory is not included in the
data tables or the statistical analysis.

Statistical Treatment

All collaborating laboratory data was evaluated statistically according to AOAC protocols using AOAC-
supplied spreadsheets. Of the 99 valid pairs of assay results reported, Laboratories 1-2, 4-5, and 7-9 had
no statistical outliers; Laboratory 6 had one statistical outlier; and Laboratory 3 had two statistical outliers.
Laboratory 4 did not report results for the canola oil plus omega-3 due to a high variability of replicates for
this matrix.

All results were evaluated for HORRAT value to determine whether the between lab reproducibility for
each matrix fell within acceptable parameters for an official AOAC method (i.e. 0.5< HORRAT value < 2).
The finite mass that is measured by this method is the mass equivalent of one hydrogen and one electron
(one hydride equivalent) which is transferred from the sample to the DPPH to form the colorless reduced
DPPH-H. Therefore, for every micromole of Trolox oxidized to reduce a micromole of DPPH, 1x10-8
grams of hydride equivalent per gram of sample are transferred to DPPH. Thus, a unit of measurement
factor of 10-8 was used for calculation of HORRAT value with the AOAC-supplied spreadsheets.

2011.XX Method for the Determination of Antioxidant Activity in Foods and Beverages by
Reaction with 2, 2’-diphenyl-1-picrylhydrazyl (DPPH)

(Applicable to the quantitative determination of total antioxidant activity in foods and beverages but not
applicable to fats and oils)

See Table 2011.XXb for the results of the interlaboratory study supporting acceptance of the method.

A. Principle

The method determines the antioxidant activity of foods by reaction with the stable radical 2,2-
diphenyl-1-picrylhydrazyl (DPPH). The DPPH free radical, which has an unpaired electron gives a strong
absorption maximum at 517 nm and is purple in color. The color turns from purple to yellow as the molar

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absorptivity of the DPPH radical at 517 nm reduces from 9660 to 1640 when the odd electron of DPPH
radical becomes paired with an electron donated from an antioxidant and a hydrogen atom (hydride
equivalent) to form the reduced DPPH-H. The resulting decolorization is stoichiometric with respect to
number of hydride equivalents captured.

Antioxidant compounds may be water-soluble, lipid-soluble, insoluble, or bound to cell walls. Thus,
extraction efficiency is an important factor in quantification of antioxidant activity of foods. This
method maximizes the extraction efficiency by adding the DPPH standard solution directly to the
sample. A separate extraction step is not performed. Antioxidant compounds are continually extracted
from the sample and immediately reacted with DPPH until the extraction & reaction is complete. The
sample is the filtered and the absorbance change is determined. Calibration is achieved as described
below by comparison to solutions of known Trolox concentration.

The sample and Trolox, a vitamin E analog which is used as the reference standard, are reacted with
DPPH solution in methanol/water for four hours at 35C in a vessel mounted on a rotary shaker 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 micromoles of Trolox equivalents (TE) per 100 grams of sample.

B. Apparatus and Materials

(a) Spectrophotometer – operating at 517 nm.

(b) Mixer/Amalgamator – Wig-L-Bug (Dentsply, MELOJEL model, www.dentsply.com).

(c) Orbital shaker – with temperature control and capacity to hold 32 x 125 mL Erlenmeyer flasks.

(d) Erlenmeyer flasks – 125 mL screw cap.

(e) Volumetric flasks (100 mL, 1 L, 2 L).

(f) Adjustable pipetter (100-1000 L) – to deliver 0.2, 0.4, 0.6, 0.8 mL.

(g) Bottle top dispenser (50 mL).

(h) Balance – readability of 0.01 mg, precision (std dev.) of  0.015 mg, capacity of 205 g.

(i) Filter paper – Whatman #4.

(j) Funnels – disposable 65 mm.

(k) Magnetic stir plate.

(l) Magnetic stir bar – 0.5 inch.

(m) Syringe filter – 25 mm with 0.45 μm nylon filter.

(n) Syringes – 10 mL with luer-loc tip.

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(o) Aluminum foil

C. Reagents:

(a) Trolox – (S)-(-)-6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Sigma Aldrich

www.sigma-aldrich.com).

(b) DPPH – 2, 2’-diphenyl-1-picrylhydrazyl (Sigma Aldrich www.sigma-aldrich.com).

(c) Methanol – HPLC grade (VWR Scientific Products www.vwrsp.com).

(d) Corn starch – Melogel (National Starch Company www.nationalstarch.com).

(e) De-ionized water ( 18.0 M-cm)

D. Reagent Solutions

(a) DPPH reagent solution. – 40 mg/L. Weigh 80.0 mg of DPPH into a plastic weigh boat.
Quantitatively transfer DPPH into a 2 L volumetric flask with 1 L HPLC grade methanol.
Surround the flask with aluminum foil to protect solution from light. Add a magnetic stir bar and
allow to stir at least 20 minutes and until all DPPH is dissolved. After DPPH is completely
dissolved add 1 L of DI water. After mixing (10-20 min), fill to volume with HPLC grade
methanol and allow to stir for a minimum of 10 minutes. Transfer to a glass bottle (2 L or greater
capacity) fitted with a dispenser capable of delivering 50 mL. Surround the bottle with aluminum
foil to protect solution from light. Add a magnetic stir bar and allow to stir until and during
transfer into sample flasks.

Note: After the addition of the DI water, the excess volume is made up with methanol to ensure
that the DPPH remains in solution. If the volume is made up with DI water, the DPPH can
precipitate out of solution.

(b) Trolox® Standard (0.5 mg/mL): Weigh 50.00 ± 0.1 mg of Trolox® into a plastic weigh boat and
record weight to 0.01 mg. Quantitatively transfer to a 100 mL low actinic volumetric flask with 50
mL HPLC grade methanol. Surround the flask with aluminum foil to protect solution from light.
Invert or stir on a magnetic stirrer until dissolved. After it is dissolved, add 50 mL DI water.
Invert 13 times and fill to volume with HPLC grade methanol.

E. Critical Control Points:

Follow manufacturer instructions and directions on all equipment. Check the dispensed volume on
pipetors and dispensettes monthly. Check calibration of balance monthly. The spectrophotometer
must run a self-diagnostic check before each day of use.

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NOTE: Calibration is the adjustment of an instrument so it provides accurate measurements. The

calibration check is a measurement of a known value by the instrument to evaluate the precision
and accuracy of the calibration of the instrument.

Glassware:
All glassware must be rinsed with HPLC grade acetone and allowed to dry after conventional
cleaning and before being used for this assay. This is to remove compounds that may interfere
with this assay. All volumetric glassware should be tested to meet its nominal value to +/- 1%
prior to use.

Deionized Water:
Deionized “DI” water used in this assay is defined as high-purity water,  18.0 M-cm.
Please see additional information in Chemicals section.

DPPH Reagent Make-up:

After the addition of the 1 liter of DI water, the excess volume is made up with methanol to ensure
that the DPPH remains in solution. If the volume is made up with DI water, the DPPH can
precipitate out of solution. Protect the DPPH Reagent from light exposure at all steps to prevent
degradation.

Trolox Standard:
Protect the Trolox standard from light exposure at all steps to prevent degradation.

F. Sample Preparation:

Dry samples must be ground to pass through a 0.5 mm sieve. Liquid samples must be homogeneous prior
to sub-sampling.

(a) Sample weigh up: A minimum of 3 subsamples is weighed for each sample (For each sample, it
is preferable that four sample weights are used such that the absorbance readings bracket the
Absorbance Target, two readings above and two below. Three values are acceptable, provided
that the values bracket the Absorbance Target (one above and two below, or vice versa). This
will give the most accurate results). The weight of each sample must be determined
experimentally for each matrix. The criterion for the correct weights for each sample for the assay
is as follows:

i. When a regression analysis is performed on the blank-corrected sample absorbances, at least

three absorbance values must be linear (R2 > 0.990) and they must bracket an absorbance
that is 50% of the DPPH standard solution as determined by the Trolox® standard curve
(typically 0.45 - 0.49 absorbance units). This value is defined as the Absorbance Target in
the Calculations section.

ii. If the sample is analyzed and no absorbance values bracket the Absorbance Target, a linear
sample curve can be used to estimate the correct range of sample weights for use in
repeating the assay so that absorbance values do bracket the Absorbance Target.

iii. If a sample is very high in antioxidants it may be necessary to dilute the sample to assure
diluted sample weights that will bracket the Absorbance Target.
i. Liquid samples can be diluted with deionized water using class A volumetric
glassware.

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ii. Solid samples are best diluted by weighing 0.1 g of sample into a Wig-L-Bug
mixing capsule and recording the actual weight to 0.0001 g, zeroing the balance
and adding 0.9 g corn starch to the capsule and recording this value to 0.0001 g.
The capsule is then mixed in the Wig-L-Bug with a small ball bearing in the
capsule to provide agitation. The dilution value is determined as the weight of the
sample divided by the weight of the sample plus the starch. Smaller sample
weights can be used to accomplish a greater dilution.

Note: The maximum number of samples per run is determined by the capacity of the orbital shaker
and by each sample being analyzed at a minimum of three different sample weights. For example,
for an orbital shaker with 32 total spots, 22 spots are available for samples (after placement of 2
blank flasks, 4 Trolox® calibration flasks, and 4 “Reference Material” flasks). By increasing the
number of analyses per sample, the chances of obtaining 3-4 acceptable absorbance values will
improve but will decrease the number of individual samples that can be analyzed per run. For the
orbital shaker specified in the Apparatus section, a maximum of 7 samples can be analyzed on a
single run.

Reference Material (RM) - A reference material made of a homogenous matrix convenient to the
lab for procurement which has been previously determined for total antioxidant activity by this
method is recommended to be run with each sample set to allow control charting to assist in
potential troubleshooting should issues arise.

(b) Label at least 3 screw cap Erlenmeyer flasks for each sample to be analyzed. Also label flasks for
2 blanks, 4 Trolox® calibration standards, and 4 Reference Materials.

Record all weights in mg to 2 decimal places.

(c) Weigh samples as described in (a) above into appropriately labeled flasks for each sample.

(d) Pipette 0.2, 0.4, 0.6 and 0.8 mL of the Trolox® standard into their respective labeled 125 mL screw
cap Erlenmeyer flasks.

(e) Add 50 mL DPPH solution (while the DPPH solution is stirring) to each Erlenmeyer flask. Swirl
to distribute sample in DPPH solution. Cap and incubate in orbital shaker at 35 ºC ± 2 ºC for 4
hours.

(f) Remove from shaker. Filter any samples that are cloudy or contain particulates through a
Whatman #4 filter paper. Some samples (such as cream soups) may require filtration through 0.45
μm syringe filter. Samples must be clear to continue with the assay. If sample is not clarified by
syringe filtration, the assay is not applicable to that sample.

(g) Read absorbances on spectrophotometer at 517 nm against a distilled water blank within 30
minutes of removal from the orbital incubator.

G. Calculations

(a) Plot Trolox® standard data: absorbance of Trolox® solutions at 517 nm (Y-axis) versus mass of
Trolox® in each solution (X-axis).

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(b) Perform a linear regression analysis on the standards data. R2 should be  0.995.
The slope (Trolox® slope) should be negative and the Y-intercept should be between 0.9-1.0
absorbance units.
The Y-intercept from this curve is the Theoretical Blank.

(c) Theoretical Blank  2 = Absorbance Target for the samples.

NOTE: Sample weights should be chosen so that a range of values bracket the Absorbance
Target. It is preferable that at least two values are above the Absorbance Target and two are
below.

(d) Tabulate data for each sample as shown in the example below (for each sample, there are
multiple flasks).

Calculate X = Corrected weight = Sample wt × Dilution factor.

Calculate Y = Net absorbance = Theoretical Blank – Absorbance (517 nm).

(e) On a separate graph for each sample, plot Net absorbance (Y-axis) versus Corrected weight (X-
axis). Perform a regression on the data. The slope should be positive. R2 should be  0.990.
For ease of calculations, it is preferable that the data are linear.

(f) Calculate Target mass = (Absorbance Target – Y-Intercept)  Slope,

where Y-intercept and Slope are obtained from the regression performed in (e) above.

(g) Calculate Total Antioxidant Activity, in mol TE / 100g,

Trolox factor  Abs target

Total Antioxidant Activity 
Target mass  Trolox slope

where TE = Trolox® equivalents,

10 6 g purity of Trolox
Trolox® factor (TF) =   100 g of sample  391546,
g m.w. of Trolox

m.w. of Trolox® = molecular weight of Trolox® = 250.29 g/mol,

purity of Trolox® for this collaborative study was 98% (0.98) as provided by the Certificate of
Analysis from the chemical manufacturer. The value for Trolox® purity can be checked by the
user by utilizing the titration method referenced in the Certificate of Analysis. The “purity of
Trolox®” used in the Trolox® factor (TF) calculation for each assay should be revised based on
the Certificate of Analysis or the individual lab’s own purity determination.

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Table 2011.XXa.   Collaborative data for determination of total antioxidant activity in foods and beverages by reaction with 2, 2’‐diphenyl‐1‐picrylhydrazyl (DPPH)
Antioxidant Activity, μmoles TE/100g
Canola Oil 
Almond Milk Blueberry Juice Carrot Juice Cocoa Green Tea Pomegranate Juice Red Wine Rosemary RTE Oat Cereal Yogurt
plus Omega‐3
Laboratory 1 7 10 20 5 13 9 18 8 19 6 16 2 11 14 21 4 12 3 15 17 22

1 144 134 1,516 1,650 7,912 6,383 153 120 119,921 116,612 530 557 3,443 3,453 1,713 1,693 92,452 103,007 1,810 1,747 495 525
2 187 226 1,846 1,818 1,958 620 224 229 133,426 130,053 733 648 3,842 4,023 1,981 1,972 107,621 103,283 1,543 1,750 496 538
a a a a
3 82 84 1,852 1,848 13,007 12,251 137 138 33,505 32,253 664 665 3,967 4,023 1,933 1,925 39,596 41,279 1,139 1,161 466 466
4 159 155 2,048 2,058 NR NR 222 188 138,600 141,200 702 712 4,286 4,363 1,937 1,952 117,700 106,300 2,203 2,057 860 885
5 ‐42 ‐7 1,994 2,025 9,798 11,080 107 128 135,624 136,635 705 695 4,091 4,485 2,047 2,100 106,890 104,071 1,662 1,825 632 630
b b
6 195 144 1,951 1,906 5,760 5,871 206 203 108,221 127,980 623 592 3,748 3,768 1,947 1,948 95,487 94,928 1,984 2,086 718 860
7 146 127 1,794 1,904 14,345 13,273 156 157 140,248 156,853 618 630 3,706 3,661 1,931 1,945 105,336 106,430 1,677 1,732 470 508
8 103 88 1,893 1,877 6,452 7,245 184 177 110,607 112,826 631 619 3,787 3,808 1,972 2,042 95,664 96,174 1,457 1,553 493 529
9 156 271 1,738 1,739 3,442 5,851 182 185 140,519 139,461 635 635 3,862 3,906 1,893 1,891 97,400 93,406 1,945 1,972 503 481

a
 Value was deleted from the data set by Single Grubb's test.
b
 Value was deleted from the data set by Cochran's test.
NR = Not Reported

Table 2011.XXb.   Statistical data from collaborative study for determination of total antioxidant activity in foods and beverages by reaction with 2, 2’‐diphenyl‐1‐picrylhydrazyl (DPPH)
Blueberry  Canola Oil plus  c Pomegranate  c RTE Oat  d
Almond Milk b Carrot Juice Cocoa Green Tea Red Wine Rosemary Yogurt
Juice Omega‐3 Juice Cereal
Statistic 1, 7 10, 20 5, 13 9,18 8, 19 6, 16 2, 11 14, 21 4, 12 3, 15 17, 22

Total number of laboratories, p 9 9 8 9 8 9 9 9 8 9 9
Total number of replicates, Sum(n(L)) 18 18 16 18 16 18 18 18 16 18 18
Overall mean of all data (grand mean), XBARBAR, μmol TE/100g 131 1,859 7,828 172 130,549 644 3,901 1,935 101,634 1,739 561
Repeatability standard deviation, s(r) 33 44 933 12 6,625 23 106 22 4,227 81 20
Reproducibility standard deviation, s(R)  76 144 4,260 38 13,873 55 289 102 7,025 302 136
Repeatability relative standard deviation, RSD(r)  25.1 2.4 11.9 7.2 5.1 3.6 2.7 1.1 4.2 4.7 3.6
Reproducibility relative standard deviation, RSD(R)  58.5 7.7 54.4 22.1 10.6 8.5 7.4 5.3 6.9 17.4 24.2
a
HORRAT Value 3.81 0.75 6.56 1.50 1.96 0.70 0.80 0.52 1.22 1.67 1.96

a ‐8
 HORRAT value was evaluated based on the finite mass of Hydride ions equivalent to the moles of Trolox reduced (i.e. 10 g hydride ions/g sample is equivalent to 1 μmole TE/100g sample)
b
 Results for Canola Oil plus Omega‐3 from laboratory 4 not received.
c
 Single Grubb's test outliers excluded.
d
 Cochran's test outliers excluded.

H. Safety

Appropriate safety glasses, lab coat, gloves and full-foot cover shoes should be worn while carrying out
all laboratory operations. Proper disposal of all chemical waste should be carried out in accordance
with Local and Federal regulations. For further detailed instructions on lab safety, please see “AOAC
Official Methods of Analysis (2000), Appendix B: Laboratory Safety”.

It should be noted that methanol used in this method is a flammable liquid and vapor. Presence of open
flames, sparks and static discharge, of heat, and oxidizing materials should be avoided. Methanol may
be fatal or cause blindness if swallowed and is harmful if inhaled or absorbed through the skin. Consult
the manufacturer’s Material Safety Data Sheet (MSDS) for complete information about all potential
hazards, appropriate precautions, proper storage and disposal.

It should also be noted that DPPH can act as a skin and respiratory sensitizer which may cause allergy
or asthma symptoms. Wear appropriate protection to avoid skin contact and to prevent inhalation.

Results and Discussion

The raw data results of the Antioxidant Activity by DPPH collaborative study are shown in Table
2011.XXa. Cochran’s and Grubbs’ outliers are noted in the table and were not used for statistical analysis.
The statistical results are shown in Table 2011.XXb. For all of the beverages tested by the participating
laboratories (almond milk, blueberry juice, carrot juice, green tea, pomegranate juice and red wine),
only almond milk did not achieve an acceptable HORRAT value. The total average Antioxidant Activity
for the almond milk was 131 µmol TE/100g was below the calculated Limit of Quantification (LOQ) of
231 µmol TE/100g. The fact that carrot juice achieved an acceptable HORRAT value of 1.50 with an

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average Antioxidant Activity of 172 µmol TE/100g suggests that the practical LOQ for the method lies
between 131 and 172 µmol TE/100g. Because no issues beyond low results were identified by any of the
laboratories with the almond milk sample and the data was distributed normally around the mean, it is the
conclusion of the Study Directors that the high HORRAT value for almond milk is a result of the average
Antioxidant Activity being less than the LOQ and not that almond milk represents an unacceptable matrix
for this assay.

The statistical results for the cocoa powder, rosemary, ready-to-eat (RTE) cereal and yogurt all gave
HORRAT values of 1.96, 1.22, 1.67 and 1.96, respectively, demonstrating that these are acceptable
matrices for analysis by this method. The Study Directors would like to point out that while the HORRAT
values for the cocoa and RTE cereal are within the acceptable range of less than 2, the variability of results
indicated by a HORRAT value greater than 1.5 may be further reduced by decreasing the sample’s particle
sizes to promote diffusion of the antioxidant compounds into the solvent system of the assay. Adherence to
the particle size specifications of this method is critical to maintaining high inter-laboratory reproducibility.
In the case of the yogurt, several laboratories reported observing issues with dispersion of the viscous dairy
product in the extraction solvent. A distribution of dispersed particle size of yogurt is likely also a function
of initial sample weight which impacts the diffusion and accessibility of the antioxidants in the matrix.

Analysis of the canola oil plus omega-3 sample showed that while reasonable within-laboratory
repeatability (RSDr) of 11.9% was observed, the inter-laboratory reproducibility (RSDR) was an
unacceptable 54.4% with a corresponding unacceptable HORRAT value result of 6.56. The oil was not
miscible in the extraction solvent which is the likely cause of the unacceptability of the matrix. Here again,
it is likely that initial sample weight is directly related in a non-linear fashion to the proportion of accessible
antioxidants at the oil/solvent interface which are available to reduce DPPH versus the amount of
antioxidant which remains sequestered within the oil droplet away from the oil/solvent interface and is thus
unavailable to reduce DPPH. Based on the results for this matrix, the Study Directors have concluded that
fats and oils are outside the matrix scope for assay by this method.

Collaborators’ Comments

One collaborator noted that at low ambient laboratory temperatures, the DPPH would partially precipitate
out of the methanol/water extraction solvent. The same collaborator also noted that they found that some
samples must be diluted first even though it is not necessary in terms of concentration. The blueberries
were cited as a case in point. Without dilution, the collaborator obtained results which were lower. The
collaborator postulated that some samples do not readily disperse their antioxidants into the methanol:
water solution. The collaborator hypothesized that, once diluted, the polyphenols may be more readily
dispersed and available for reaction with DPPH.

Another collaborator commented that they realized that for all juice samples the method works quite
consistently but for oily samples and powders it was quite difficult for them to find the correct value for the
sample weight.

A third collaborator commented that in making the DPPH reagent, it was difficult to see if there were any
undissolved crystals in the bulk solvent. The collaborator solvated the DPPH in a 250 mL beaker with
stirring and after 30 minutes, filtered through a glass stem funnel into the volumetric flask. No undissolved
crystals were observed. This collaborator also commented that they had trouble in zeroing in on
appropriate starting weights due to unfamiliarity with the blind samples provided. The cocoa and rosemary
samples were particular difficult for this collaborator. They had to dilute these solid samples using Melojel
and mixing in plastic centrifuge tubes.

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Recommendation

The Study Directors recommend that this method be adopted by AOAC INTERNATIONAL as Official
First Action for all foods and beverages excepting fats and oils.

Acknowledgements

This study was supported by funding from Pom Wonderful, Inc., Del Rey, California, USA. The Study
Directors would like to thank the following collaborators for all of their efforts in completing this study:

Alla Begelman, Medallion Laboratories, Minneapolis, MN

Mary Camire, University of Maine, Orono, ME
Michael P. Doherty, University of Maine, Orono, ME
Christopher DeRito, Cornell University, Ithaca, NY
Matthew Hanson, General Mills, Inc., Minneapolis, MN
Michael Marquardt, General Mills, Inc., Minneapolis, MN
Jonathan W. DeVries, Medallion Labs/General Mills, Inc., Minneapolis, MN
Ronghua Liu, Guelph Food Research Centre, Guelph, Ontario, Canada

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