1562  Plank et al.: Journal of AOAC International Vol. 95, No.

6, 2012

ANTIOXIDANTS

Determination of Antioxidant Activity in Foods and Beverages

by Reaction with 2,2′-Diphenyl-1-Picrylhydrazyl (DPPH):
Collaborative Study First Action 2012.04
David W. Plank
Medallion Laboratories, 9000 Plymouth Ave North, Minneapolis, MN 55427
John Szpylka
General Mills, Inc., 9000 Plymouth Ave North, Minneapolis, MN 55427
Harry Sapirstein
University of Manitoba, Department of Food Science and Nutrition, Winnipeg, Manitoba, MB R3T 2N2, Canada
David Woollard
New Zealand Laboratory Services Ltd, 35 O’Rorke Rd, Penrose, Auckland 1642, New Zealand
Charles M. Zapf
McCormick & Co., Inc., Technical Innovation Center, 202 Wight Ave, Hunt Valley, MD 21031
Vong Lee
POM Wonderful, LLC, 5286 S. Del Rey Ave, Del Rey, CA 93616
C-Y. Oliver Chen
Tufts University, Jean Mayer USDA Human Nutrition Research Center on Aging, 711 Washington St, Boston, MA 02111
Rui Hai Liu
Cornell University, Department of Food Science, 108 Stocking Hall, Ithaca, NY 14853
Rong Tsao
Guelph Food Research Centre, Agriculture and Agri-Food Canada, 93 Stone Rd West, Guelph, Ontario N1G 5C9, Canada
André Düsterloh
DSM Nutritional Products, Ltd, Bldg 205, Rm 6, Wurmisweg 576, 4303 Kaiseraugst, Switzerland
Steve Baugh
AOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Gaithersburg, MD 20877

Collaborators: A. Begelman; M. Camire; C. DeRito; J.W. DeVries; M.P. Dougherty; M. Hanson; R. Liu; M. Marquard; A. Ser;
M. Stringer

A colorimetric method for the determination of
total antioxidant activity in a variety of foods and
beverages was validated in both a single-laboratory
validation and a collaborative laboratory validation
study. The procedure involved 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 was monitored by spectrophotometric
measurement of the absorbance loss at 517 nm.
Antioxidant activity was quantified relative to
a dilution series of vitamin E analog standards
(Trolox®), which were analyzed in parallel
simultaneously with the food and beverage samples.
The antioxidant activities of the samples ranged
from 131 to 131 000 µmole Trolox equivalents/100 g.
Statistical analysis of the results showed that nine
of the 11 matrixes gave acceptable HorRat values,
indicating that the method performed well in these
cases. The acceptable matrixes include pomegranate
juice, blueberry juice, carrot juice, green tea, wine,
rosemary spice, ready-to-eat cereal, and yogurt.
Two samples failed the HorRat test: the first was
an almond milk that had an antioxidant level below
the practical LOQ for the method; the second was
a sample of canola oil with added omega-3 fatty
acid that was immiscible in the reaction medium.

Submitted for publication July 24, 2012.
The method was approved by the Expert Review Panel on Strategic
Foods Analytical Methods as First Action. See “Standards News,”
(2012) Inside Laboratory Management, March/April issue.
The AOAC Stakeholder Panel on Strategic Foods Analytical
Methods (SPSFAM) invites method users to provide feedback on the
First Action methods. Feedback from method users will help verify
that the methods are fit for purpose and are critical to gaining global
recognition and acceptance of the methods. Comments can be sent
directly to the corresponding author.
Corresponding author’s e-mail: david.plank@medlabs.com
DOI: 10.5740/jaoacint.CS2012_04
O
n March 28, 2011, the AOAC Board of Directors approved
an alternative path to achieve Official First Action status
for methods selected. Following this process, selected
methods are reviewed and approved by an expert review panel
(ERP) for AOAC Official First Action status. This process
follows selected methods for a period of approximately 2 years
as First Action methods, allowing an opportunity for methods to
be used in laboratories and to generate additional information.
The ERP will monitor the method’s performance, and after about
 Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012  1563
2  years, will recommend the method to the Official Methods
Board for Final Action if the method is found to be suitable (1).
The ERP on Strategic Foods Analytical Methods reviewed
the method “Determination of Antioxidant Activity in Foods
and Beverages by Reaction with 2,2′-Diphenyl-1-Picrylhydrazyl
(DPPH)” during the AOAC INTERNATIONAL Mid-Year
Meeting on March 22, 2012. After evaluating the data available,
the ERP agreed that the method meets standard method
performance requirements, as articulated by the Stakeholder
Panel on Strategic Foods Analytical Methods. The ERP granted
the method First Action status, applicable to antioxidant testing in
foods and beverages.
The stakeholder panel recognized that the data for antioxidant
activity are used primarily as a marketing tool. These values are
obtained from in vitro measurements and cannot necessarily be
extrapolated to in vivo activities.
The stakeholder panel also recognized the need for validated
antioxidant methods to allow manufacturers a common basis
for comparison. However, after considerable deliberation, the
ERP determined that each of the different antioxidant activity
methods provides an independent, nonequivalent, indication
of activity. Thus, DPPH methods provide DPPH values just as
oxygen radical absorbance capacity (ORAC) methods provide
ORAC values. Both are indicators of antioxidant activity, neither
are necessarily a true measure of in vivo antioxidant activity, and
the two are not equivalent for all compositions of matrixes. This
is also true for other methods that propose to measure antioxidant
activity. The ERP determined that the crucial delimiter of the
scope of the method is the measurement process, and it must be
included in the title of the method. Thus, a direct comparison of
absolute values between methods is not valid. Comparison of the
absolute values for antioxidant activity in different foods is only
valid if the same method is used.
Antioxidant compounds in foods 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 (2–6).
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.
Because it is impossible or impractical to measure each
antioxidant in foods or biological samples, 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, or spectrophotometry. Early methods
often required 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. Other methods
determine the resistance of lipid or lipid emulsions to oxidation in
the presence of the antioxidant being tested. Malondialdehyde or
thiobarbituric acid-reactive-substances (7) assays have been used
extensively since the 1950s 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
that 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. 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 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 and inhibition of the free radical action is
measured (10) using the fluorescent compound, B-phycoerythrin,
R-phycoerythrin, or fluorescein. Vinson et al.  (11) measured
phenolics, a significant class of plant-based antioxidants, in fruits
and vegetables using the Folin-Ciocalteu colorimetric reagent
with inhibition of low-density lipoprotein oxidation mediated by
cupric ions.
The use of the free radical 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 analog [Trolox (S)-(-)-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid] is the usual way to
express results, i.e., µmole Trolox equivalents (TE)/100 g. In this
study, the complete sample is continuously reacted with DPPH
in methanol–water for 4 h 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 has been characterized  (12,  13). DPPH
is reduced by the antioxidants through the transfer of a proton
plus an 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 that each of these methods measures 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 that comparisons between labels are valid.
This study has been undertaken to establish the accuracy, between-
1564  Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012

Table  1.  Results of precollaborative ruggedness testing collaborative study for analysis as blind duplicates by the
(µmole TE/100 g reported by laboratories) participating laboratories. The matrixes ranged from a variety of
beverages (almond milk, blueberry juice, carrot juice, green tea,
Sample/sample No.
pomegranate juice, and red wine) to fermented dairy (yogurt) to
Pomegranate juice Oat cereal base ground solids (rosemary and ready-to-eat cereal) to oils (canola
Lab No. K31610 P31510 RM25F oil plus omega-3).
a
All samples were coded and randomized to ensure the blind
1 3533 4368 2785
nature of the duplicates for each matrix as shipped by the Study
2 3768 4314 2608 Directors. The samples were all prepared to a homogeneous
3 3566 3673 1777 distribution prior to subsampling. The samples were packed in
4 3312 3398 1570 individual 40 mL glass vials and sealed with septa. An argon flush
5 3504 3610 2181
in the headspace above each sample was performed, and an argon
blanket was left over each sample. Samples were shipped to each
6 3201 3264 1659
laboratory by overnight air with instructions to transfer samples
7 3900 4000 2000 to 4°C storage conditions in the dark on arrival.
8 3624 3683 2353 A total of nine laboratories reported data for the collaborative
9 3564 3776 NR
b study samples. An additional laboratory had to withdraw due
Avg. 3552 3787 2021 to samples arriving while the principal investigator was not
available. Samples sat at room temperature for a period of
SR 211 378 382
3  weeks prior to analysis. Clear indications of aberrant results
RSDR, % 5.94 9.97 18.89
for the samples led the laboratory to request withdrawal from the
HorRat valuec 0.64 1.08 1.86 study. Data from the withdrawn laboratory is not included in the
a
 Statistical outlier. data tables or the statistical analysis.
b
 NR = Not reported.
c Statistical Treatment
 HorRat value was elevated based on the finite mass of hydride ions
equivalent to the moles of Trolox oxidized per mole DPPH reduced
–8
(i.e., 10 g hydride ions/g sample is equivalent to 1 µmole TE/100 g All collaborating laboratory data were evaluated statistically
sample).
according to AOAC protocols using AOAC-supplied spreadsheets.
Of the 99 valid pairs of assay results reported, Laboratories 1, 2,
laboratory reproducibility, and utility of the DPPH antioxidant 4, 5, 7, and 9 had no statistical outliers; Laboratory 6 had one
method as a reliable, standardized method for assessing the statistical outlier; and Laboratory 3 had two statistical outliers.
antioxidant capacity of foods and beverages. Laboratory 4 did not report results for the canola oil plus omega-3
due to a high variability of replicates for this matrix.
Collaborative Method Validation Study All results were evaluated for HorRat value to determine
whether the between-laboratory reproducibility for each matrix
fell within acceptable parameters for an official AOAC method,
Precollaborative Ruggedness Study
i.e., 0.5 < HorRat value ≤2. The finite mass that is measured by
A precollaborative ruggedness study was conducted with this method is the mass equivalent of one hydrogen and one
the participating laboratories to ensure adequate method electron (one hydride equivalent), which is transferred from the
performance. Laboratories participating in the evaluation were sample to the DPPH to form the colorless reduced DPPH-H.
sent three samples packed under an argon layer and sealed by Therefore, for every micromole of Trolox oxidized to reduce a
septa along with copies of the method, and 1  g each of DPPH micromole of DPPH, 1 × 10–8 g of hydride equivalent/g of sample
and Trolox. Each laboratory was requested to run each sample is transferred to DPPH. Thus, a unit of measurement factor of
in singlet. Laboratories were requested to conduct the analysis, 10–8 was used for calculation of HorRat value with the AOAC-
ask questions regarding procedures, and provide feedback to the supplied spreadsheets.
Study Directors on any aspects of the method about which the
collaborator might have a concern. The results of the analyses on AOAC Official Method 2012.04
the ruggedness testing samples are shown in Table 1. Relevant Antioxidant Activity in Foods and Beverages
comments received from the participating laboratories were by Reaction with 2,2′-Diphenyl-1-Picrylhydrazyl
incorporated as changes to improve the method as appropriate. (DPPH)
No significant procedural changes were found to be necessary. First Action 2012
The results for the precollaborative ruggedness study gave
a HorRat value for all samples greater than 0.5 but less than 2, [Applicable to the determination of antioxidants in foods
which indicates that the laboratories’ analysis results meet the and beverages by reaction with 2,2′-diphenyl-1-picrylhydrazyl
criteria for acceptable method performance in accordance with (DPPH).]
AOAC guidance (19–21). See Tables 2012.04A and B for results of the interlaboratory
study supporting acceptance of the method.
Collaborative Study Protocol
A.  Principle
Eleven food samples representing products commonly
marketed for their antioxidant activities were selected for the The method determines the antioxidant activity of foods and
Table  2012.04A.  Collaborative results for determiniation of total antioxidant activity in foods and beverages by reaction with 2,2′-diphenyl-picrylhydrazyl (DPPH)
Antioxidant activity, µmole TE/100 g

Canola oil plus

Almond milk Blueberry juice omega-3 Carrot juice Cocoa Green tea Pomegranate juice Red wine Rosemary RTE oat cereal Yogurt

Lab R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2

1 144 134 1516 1650 7912 6383 153 120 119921 116612 530 557 3443 3453 1713 1693 92452 103007 1810 1747 495 525
a
2 187 226 1846 1818 1958 620 224 229 133426 130053 733 648a 3842 4023 1981 1972 107621 103283 1543 1750 496 538
b b
3 82 84 1852 1848 13007 12251 137 138 33505 322533 664 665 3967 4023 1933 1925 39596b 41279b 1139 1161 466 466
c
4 159 155 2048 2058 NR NR 222 188 138600 141200 702 712 4286 4363 1937 1952 117700 106300 2203 2057 860 885
a
5 –42 –7 1994 2025 9798 11080 107 128 135624 136635 705 695 4091 4485a 2047 2100 106890 104071 1662 1825 632 630
a
6 195 144 1951 1906 5760 5871 206 203 108221 127980 623 592 3748 3768 1947 1948 95487 94928 1984 2086 718 860a
7 146 127 1794 1904 14345 13273 156 157 140248 156853 618 630 3706 3661 1931 1945 105336 106430 1677 1732 470 508
8 103 88 1893 1877 6452 7245 184 177 110607 112826 631 619 3787 3808 1972 2042 95664 96174 1457 1553 493 529
9 156 271 1738 1739 3442 5851 182 185 140519 139461 635 635 3862 3906 1893 1891 97400 93406 1945 1972 503 481
a
 Value was deleted from the data set by Cochran’s test.
b
 Value was deleted from the data set by single Grubbs’ test.
c
 NR = Not reported.

Table  2012.04B.  Statistical data from collaborative study for determination of total antioxidant activity in foods and beverages by reaction with 2,2′-diphenyl-1-picrylhydrazyl
(DPPH)
Almond Blueberry Canola oil plus Pomegranate RTE oat
a
milk juice omega-3 Carrot juice Cocoab Green tea juice Red wine Rosemaryb cereal Yogurtc

Statistic 1, 7 10, 20 5, 13 9, 18 8, 19 6, 16 2, 11 14, 21 4, 12 3, 15 17, 22

Total No. of labs, p 9 9 8 9 8 9 9 9 8 9 8

Total No. replicates, sum(n(L)) 18 18 16 18 16 18 18 18 16 18 16

Overall mean of all data (grand mean), x, µmole TE/100 g 131 1859 7828 172 130549 644 3901 1935 101634 1739 561
Repeatability standard deviation, s(r) 33 44 933 12 6625 23 106 22 4227 81 20
Reproducibility standard deviation, s(R) 76 144 4260 38 13873 55 289 102 7025 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
HorRat valued 3.81 0.75 6.56 1.50 1.96 0.70 0.80 0.52 1.22 1.67 1.96
a
 Results for canola oil plus omega-3 from Laboratory 4 not received.
b
 Single Grubbs’ test outliers excluded.
c
 Cochran’s test outliers excluded.
d
 HorRat value was evaluated based on the finite mass of hydride ions equivalent to the moles of Trolox reduced (i.e., 10–8 g hydridge ions/g sample is equivalent to 1 µmole TE/100 g sample).
Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012  1565
1566  Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012

Table  2012.04C.  Results of single-laboratory method precision study (µmole TE/100 g reported by laboratory)
Pomegranate

Day/replicate Cocoa RTE cereal Blueberry juice Green tea Carrot juice Red wine Juice Concentrate By-product Arils

1/1 119392 3107 1795 822 379 3093 3492 9938 199985 16188
a
1/2 117390 3147 1938 843 394 3031 3512 9826 256657 17713
1/3 112408 3234 1943 826 390 2842 3525 10212 204535 17090
2/1 120003 3049 1917 803 375 2961 3664 10101 216800 19145
2/2 121937 3087 1921 810 372 3268 3617 10075 208899 17327
2/3 114366 3038 1985 803 350 3089 3703 9910 204339 17821
3/1 127784 3093 1938 845 371 2943 3449 9718 197172 18680
3/2 127446 3071 1893 849 378 3074 3071 9750 185353 17278
3/3 119581 3085 1927 840 381 3162 3386 9854 188794 18184
Avg. 120034 3101 1917 827 377 3051 3491 9932 200735 17714
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
HorRat valueb 0.79 0.20 0.27 0.19 0.26 0.43 0.57 0.21 1.01 0.68
a
 Statistical outlier.
 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
b

hydride ions/g sample is equivalent to 1 µmole TE/100 g sample).

beverages by reaction with the stable radical DPPH. This method (c)  Orbital shaker.—With temperature control and capacity to
is not applicable to high-fat and -oil matrixes (>50% fat). The hold 32 × 125 mL Erlenmeyer flasks.
DPPH free radical gives a strong absorption maximum at 517 nm (d)  Erlenmeyer flasks.—125 mL screw-cap.
and is purple in color. The color turns from purple to yellow as the (e)  Volumetric flasks (100 mL, 1 L, and 2 L).
molar absorptivity of the DPPH radical at 517 nm decreases from (f)  Adjustable pipettor (100–1000 µL).—To deliver 0.2, 0.4,
–1 –1 0.6, and 0.8 mL.
9660 to 1640 mM cm when the odd electron of DPPH radical
becomes paired with an electron donated from an antioxidant (g)  Bottle top dispenser (50 mL).
and a hydrogen atom (hydride equivalent) to form the reduced (h)  Balance.—Readability of 0.01 mg, precision (SD) of
DPPH-H. The resulting decolorization is stoichiometric with ±0.015 mg, capacity of 205 g.
respect to number of hydride equivalents captured. (i)  Filter paper.—Whatman No. 4.
Antioxidant compounds may be water-soluble, lipid-soluble, (j)  Funnels.—Disposable, 65 mm.
insoluble, or bound to cell walls. Thus, extraction efficiency is (k)  Magnetic stir plate.
an important factor in the quantification of antioxidant activity of (l)  Magnetic stir bar.—0.5 in.
foods. This method maximizes the extraction efficiency by adding (m)  Syringe filter.—25 mm with 0.45 μm nylon filter.
(n)  Syringes.—10 mL with Luer-lock tip.
the DPPH standard solution directly to the sample. A separate
(o)  Aluminum foil.
extraction step is not performed. Antioxidant compounds are
continually extracted from the sample and immediately reacted
C.  Chemicals
with DPPH until the extraction and reaction are complete. The
sample is then filtered, and the absorbance change is determined.
(a)  Trolox.—(S)-(-)-6-hydroxy-2,5,7,8-tetramethyl- chroman-
Calibration is achieved as described below by comparison to 2-carboxylic acid (Sigma-Aldrich, www.sigma-aldrich.com).
solutions of known Trolox concentration. (b)  DPPH.—2,2′-Diphenyl-1-picrylhydrazyl (Sigma-
The sample and Trolox, a vitamin E analog which is used Aldrich).
as the reference standard, are reacted with DPPH solution in (c)  Methanol.—HPLC grade (VWR Scientific Products,
methanol–water for 4 h at 35°C in a vessel mounted on a rotary www.vwrsp.com).
shaker, and the absorbance changes are measured at 517 nm. The (d)  Corn starch.—Melogel (National Starch Co., www.
quantity of sample necessary to react with one-half of the DPPH nationalstarch.com).
is expressed in terms of the relative amount of Trolox reacted. (e)  Deionized (DI) water (≥18.0 MΩ-cm).
Antioxidant activity of a sample is expressed in terms of µmole
Trolox equivalents (TE)/100 g sample. D.  Reagent Solutions

B.  Apparatus and Materials Appropriate safety glasses, laboratory coat, gloves, and full-
foot cover shoes should be worn while carrying out all laboratory
(a)  Spectrophotometer.—Operating at 517 nm. operations. Proper disposal of all chemical waste should be carried
(b)  Mixer/amalgamator.—Wig-L-Bug (Dentsply, MELOJEL out in accordance with local and federal regulations. For further
model, www.dentsply.com). detailed instructions on laboratory safety, see Official Methods
 Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012  1567
of Analysis of AOAC INTERNATIONAL (2000), Appendix  B:
Laboratory Safety (22).
Methanol used in this method is a flammable liquid and vapor.
The presence of open flames, sparks and static discharge, 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 for complete information about all potential
hazards, appropriate precautions, proper storage, and disposal.
Because 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.
(a)  DPPH reagent solution.—40 mg/L. Weigh 80.0 mg 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 min and until all
DPPH is dissolved. After DPPH is completely dissolved, add 1 L
DI water. After mixing (10–20 min), fill to volume with HPLC
grade methanol and stir for a minimum of 10  min. 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, or alternatively use low actinic
glassware. Add a magnetic stir bar and allow to stir until and
during transfer into sample flasks. Note: After 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
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’s instructions and directions on all
equipment. Check the dispensed volume on pipettors and
dispensets monthly. Check calibration of balance monthly. The
spectrophotometer must run a self-diagnostic check before each
day of use. Note: Calibration is the adjustment of an instrument
so that 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 instrument.
(a)  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.
(b)  DI water.—DI water used in this assay is defined as
high-purity water, ≥18.0 MΩ-cm. See additional information in
Chemicals section.
(c)  DPPH reagent make-up.—After addition of 1 L 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.
Make up DPPH reagent solution the day of analysis for each
sample set.
(d)  Trolox standard.—Protect the Trolox standard from light
exposure at all steps to prevent degradation. Trolox standard may
be stored at 4°C for up to 2 weeks.
(e)  Particle size control.—Method uncertainty and
interlaboratory variability 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 interlaboratory reproducibility.
F.  Sample Preparation
Dry samples must be ground to pass through a 0.5 mm sieve.
Liquid samples must be homogeneous prior to subsampling.
Sample weigh up.—A minimum of three subsamples are
weighed for each sample. For each sample, it is preferable to use
four sample weights 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:
When a regression analysis is performed on the blank-
corrected sample absorbances, at least three absorbance values
2
must be linear (R > 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 below.
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. If a
sample is very high in antioxidants, it may be necessary to dilute
the sample to ensure diluted sample weights that will bracket the
absorbance target.
Liquid samples can be diluted with DI water using class A
volumetric glassware.
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 two blank flasks, four
Trolox calibration flasks, and four reference material flasks).
By increasing the number of analyses per sample, the chances
of obtaining three to four 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
1568  Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012
Apparatus section, a maximum of seven samples can be analyzed
in a single run.
Reference material.—A reference material, made of a
homogeneous matrix convenient to the laboratory for procurement
and 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.
Label at least three screw-cap Erlenmeyer flasks for each
sample to be analyzed. Also label flasks for two blanks, four
Trolox calibration standards, and four reference materials. Record
all weights in mg to two decimal places.
Weigh samples as described in the Sample weigh up section.
Pipet 0.2, 0.4, 0.6, and 0.8 mL of the Trolox standard into
their respective labeled 125  mL screw-cap Erlenmeyer flasks.
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 ± 2°C for 4 h
at 250 rpm.
Remove from shaker. Filter any samples that are cloudy or
contain particulates through a Whatman No. 4 filter paper. Some
samples (such as cream soups) may require filtration through a
0.45 μm syringe filter. Samples must be clear to continue with the
assay. If a sample is not clarified by syringe filtration, the assay is
not applicable to that sample.
Read absorbances on spectrophotometer at 517 nm against a
distilled water blank within 30 min of removal from the orbital
incubator.
G.  Calculations
Plot Trolox standard data: absorbance of Trolox solutions at
517 nm (Y-axis) versus mass of Trolox in each solution (X-axis).
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.
Theoretical blank ÷ 2 = absorbance target for the samples
Note: Sample weights should be chosen so that a range of
values brackets the absorbance target. It is preferable that at least
two values are above the absorbance target and two are below.
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)
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.
Calculate target mass =
(absorbance target – Y-intercept) ÷ slope
where Y-intercept and slope are obtained from the regression plot
of the data.
Calculate total antioxidant activity, in µmole TE/100 g
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 mw of Trolox
mw of Trolox = 250.29 g / mol
The purity of Trolox for this collaborative study was 98%,
as provided by the Certificate of Analysis from the chemical
manufacturer. The user can check the value for Trolox purity
by utilizing the titration method referenced in the Certificate of
Analysis. The “purity of Trolox” used in the TF calculation for
each assay should be revised based on the Certificate of Analysis
or the individual laboratory’s own purity determination.
H.  Method Validation
(a)  Single-laboratory validation study.—In advance of the
collaborative study, a single-laboratory validation was performed
to assess the method’s precision and instrument response range.
(b)  Method precision.—The precision of the method was
assessed on 10 matrixes. Each matrix was analyzed in triplicate
on each of 3 separate days, thereby obtaining nine antioxidant
activity results for each matrix. This was performed by two
technicians each analyzing all 10 matrixes at separate times. The
precision study is summarized in Table 2012.04C and adequately
demonstrates the reliable repeatability of this method for a variety
of food matrixes.
(c)  Linearity and instrument response range.—A Trolox
standard solution (50.00 mg in 100 mL 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.
(d)  Calculation of method LOQ.—Calculation of the method
LOQ was performed using the two equations described in the
method calculations. Combining both equations yields:
Total antioxidant activity =
[(391546) × (absorbance target)]/[(target mass) × (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] × [(391546  µmole TE/100  g Trolox)
× (1/2 × 0.911 abs)]/[(0.5 g sample) × (1.5415 abs/mg Trolox)]
= 2.31 µmole TE/g sample or 231 µmole TE/100 g sample.
Reference: J. AOAC Int. 95, 1562(2012)
Results and Discussion
The raw data results of the antioxidant activity by DPPH
collaborative study are shown in Table 2012.04A. The outliers
of Cochran and Grubbs are noted in the table and were not
 Plank et al.: Journal of AOAC International Vol. 95, No. 6, 2012  1569
used for statistical analysis. The statistical results are shown in
Table 2012.04B. 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 µmole TE/100 g, and was
below the calculated LOQ of 231 µmole TE/100 g. The fact that
carrot juice achieved an acceptable HorRat value of 1.50 with
an average antioxidant activity of 172 µmole TE/100 g suggests
that the practical LOQ for the method lies between 131 and
172 µmole TE/100 g. Because no issues beyond low results were
identified by any of the laboratories with the almond milk sample
and the data were distributed normally around the mean, the
Study Directors concluded 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 cereal, and yogurt gave HorRat values of 1.96,
1.22, 1.67, and 1.96, respectively, demonstrating that these are
acceptable matrixes for analysis by this method. The Study
Directors point out that while the HorRat values for the cocoa
and ready-to-eat cereal are within the acceptable range of <2,
the variability of results indicated by a HorRat value >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 interlaboratory
reproducibility. In the case of the yogurt, several laboratories
reported issues with dispersion of the viscous dairy product in
the extraction solvent. A distribution of dispersed particle size of
yogurt is likely 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 a reasonable within-laboratory repeatability (RSDr) of
11.9% was observed, the interlaboratory 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 probable that initial sample weight
is directly related in a nonlinear fashion to the proportion of
accessible antioxidants at the oil/solvent interface that 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.
Acknowledgments
This study was supported by funding from Pom Wonderful,
Inc., Del Rey, CA. 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
Christopher DeRito, Cornell University, Ithaca, NY
Jonathan W. DeVries, Medallion Laboratories/General Mills,
Minneapolis, MN
Michael P. Dougherty, University of Maine, Orono, ME
Matthew Hanson and Michael Marquard, General Mills,
Minneapolis, MN
Ronghua Liu, Guelph Food Research Centre, Guelph, Ontario,
Canada
Alison Ser and Michael Stringer, University of Manitoba,
Winnepeg, Manitoba, Canada
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