1466  Kupina et al.: Journal of AOAC International Vol. 101, No.
5, 2018
Dietary Supplements AND TRADITIONAL MEDICINE
Determination of Total Phenolic Content Using the Folin-C
Assay: Single-Laboratory Validation, First Action 2017.13
Steve Kupina
Polyphenolics, a division of Constellation Brands, 12667 Rd 24, Madera, CA 93637
Chris Fields
Applied Foods Sciences, LLC, 1700 S. Lamar Blvd, Austin, TX 78704
Mark C. Roman1
Tampa Bay Analytical Research, Inc., 10810 72nd St, Suite 206, Largo, FL 33777
Sharon L. Brunelle
Brunelle Biotech Consulting, 6620 NW Burgundy Dr, Corvallis, OR 97330
A single-laboratory validation of a method using Folin &
Ciocalteu’s phenol reagent (Folin-C reagent) for
determination of total phenolic content of selected
dietary supplement extracts was performed. The
method is composed of a water extraction of dried
extracts with sonication followed by reaction with
the Folin-C reagent. The resulting colorimetric
reaction is measured at 765 nm and compared with
a standard curve generated with gallic acid standard
solutions. The validation results were compared with
Standard Method Performance Requirement (SMPR®)
2015.009, developed by the Stakeholder Panel on
Dietary Supplements. The method demonstrated
acceptable within-day RSDr of 1.96–7.47% for the
five matrixes studied (grape seed extract, grape skin
extract, black tea extract, green coffee extract, and
cocoa extract). When gallic acid was spiked into
maltodextrin (a surrogate dietary supplement carrier)
at 30 or 70%, the recovery ranged from 91 to 104%,
within the acceptable range established by SMPR
2015.009. Selectivity testing with glucose, fructose,
and sucrose demonstrated no positive interference
by these compounds. Finally, ruggedness studies
demonstrated no significant effects due to changes in
the heating apparatus, test material weight, read time
after reaction, amount of Folin-C reagent, reaction
time, reaction temperature, and amount of Na2CO3. The
single-laboratory validation results support adoption
of the method as First Action Official Method SM
2017.13 and further evaluation in a collaborative study.
Received February 6, 2018. Accepted by PB April 12, 2018.
This method was approved by the AOAC Expert Review Panel for
Dietary Supplements as First Action.
The Expert Review Panel Folin-C 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 is critical for
gaining global recognition and acceptance of the methods. Comments
can be sent directly to the corresponding author or methodfeedback@
aoac.org.
1
Deceased.
Corresponding author’s e-mail: steve.kupina@cbrands.com
DOI: https://doi.org/10.5740/jaoacint.18-0031
P
olyphenols are found in fruits, vegetables, cereals, and
beverages and help defend against the harmful effects
of ultraviolet radiation (1). The antioxidant property of
polyphenols in the diet over a long period has been linked to
protection against development of cancers, cardiovascular dis­
eases, diabetes, osteoporosis, and neurodegenerative diseases (2).
Dietary supplements consisting of extracts of plant material
rich in phenolic compounds are now available, and their health
benefits and biochemical effects are being studied (3). To aid
in the interpretation of these studies and to provide consumer
protections, a method is needed to quantify the phenolic content
of dietary supplements. This paper describes the validation
of a method based on the reaction of phenolic compounds
with the Folin-Ciocalteu reagent, a complex mixture of
heteropolyphosphotungstate-molybdate, in the presence of
sodium carbonate. The method is based on earlier work by
Singleton and Rossi (4).
AOAC Official Method 2017.13
Total Phenolic Content in Extracts
Folin-C (Folin & Ciocalteu) Colorimetric Method
First Action 2017
[Applicable to the determination of total polyphenolic
content, expressed as gallic acid equivalents, in grape seed,
grape skin, coffee, cocoa, and black tea extracts with >5% total
phenolic content.]
A. Principle
Phenols are first extracted in water and then reacted with
the Folin-C reagent (a complex mixture of heteropoly­
phosphotungstate-molybdate) in the presence of sodium
carbonate to form a blue-colored complex. The intensity
of the blue color is proportional to the amount of reactive
phenolic compounds in the sample. The phenolic content
is determined by measuring the absorbance of the sample
solution at 765 nm and comparing it with a calibration
curve using gallic acid as a standard. The method is able
to quantify total polyphenolic content of about 5 (w/w) to
100% (w/w) in the extracts. The method will also yield
positive results for extracts containing ascorbic acid, amino
acids, or sugars.
 Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018  1467

B. Apparatus Table 2017.13.  Preparation of calibration standard

solutions
Note: Equivalent apparatus may be substituted. All
volumetric pipettes and volumetric flasks are Class A. Calibration
(a)  UV-Vis spectrophometer.—Capable of measuring standard Volume of stock Volume of Approximate concn
solution standard solution, mL flask, mL of gallic acid, mg/L
absorbance at 765 nm in 1 cm quartz cuvettes.
(b)  Analytical balance.—Accurate to ±0.01 mg. 1 1 25 40
(c)  Test tubes.—25 × 150 mm Pyrex rimless (VWR Cat. No. 2 2 25 80
60820-320, Corning 9820).
3 3 25 120
(d)  Volumetric flasks.—50, 100, and 1000 mL.
4 4 25 160
(e)  Volumetric pipettes.—1, 2, 3, 4, 5, and 15 mL.
(f)  Adjustable pipettor.—1–5 mL. 5 5 25 200
(g)  Bottles.—1, 3, and 15 mL with bottle top dispensers.
(h)  Vortex mixer.
E. Determination
(i)  Dry block heater.—Capable of maintaining 30 ± 2°C and
accepting 25 × 150 mm tubes.
(a)  Colorimetric reaction.—Prepare a series of seven test
(j)  Ultrasonic bath.
tubes, each containing 15.00 mL water and 1 mL Folin &
(k)  Ice water bath.
Ciocalteu’s phenol reagent. Into each test tube, add one of
(l)  Cuvettes.—Quartz, 1 cm.
the following: (1) 1.00 mL sample test solution; (2) 1.00 mL
C. Reagents calibration standard solution 1–5; or (3) 1.00 mL water (blank).
Mix the contents of each test tube well and allow to sit for 6 min.
(a)  Water.—HPLC grade. Add 3.0 mL sodium carbonate solution to each tube and mix
(b)  Folin & Ciocalteu’s phenol reagent.—Cat. No. F9252, well. Place the test tubes in the heating block for 120 min.
Sigma-Aldrich (St. Louis, MO). Note: Formulations of (b)  Measurement.—In duplicate, transfer 1 mL of each
Folin & Ciocalteu phenol reagent vary among manufacturers, reaction to a cuvette and measure the absorbance at 765 nm after
and method performance could be compromised if the zeroing the spectrophotometer with blank. Measure calibration
formulation varies from the prescribed product. standard solutions first, then sample test solutions. Report mean
(c)  Sodium carbonate.—Anhydrous, Cat. No. 7527, of duplicate measurements.
Mallinckrodt (Phillipsburg, NJ), or equivalent.
(d)  Sodium carbonate solution (20% Na2CO3).—Transfer F. Calculations
200 g sodium carbonate into a 1000 mL volumetric flask. Fill to
volume with water and mix well. (a)  Stock standard solution.—The concentration of gallic
(e)  Gallic acid.—Crystalline, ≥98%, Cat. No. G7384, Sigma- acid in the stock standard solution is calculated as follows:
Aldrich, or equivalent. Store desiccated at room temperature. Stock standard solution (mg/L) = M × P × (1 – W ) × 1000

D.  Preparation of Test Solutions
(a)  Preparation of calibration standard solutions.—
Accurately weigh about 1.1 (±0.1) g gallic acid and transfer
into a 1000 mL volumetric flask (record weight to nearest
0.0001 g). Add about 750 mL water and sonicate in an ultrasonic
bath containing about 3 cm water for up to 10 min until the
solids are dissolved. Dilute to volume with water and mix well.
Label as “stock standard solution.” This stock standard solution
(ca 1000 mg/L gallic acid) may be used for up to 1 month
when stored at 2–8°C. Prepare calibration standard solutions
as presented in Table 2017.13 (ca 40–200 mg/L gallic acid) by
pipetting the indicated amount of stock standard solution into
the indicated size volume flask and diluting to volume with
water. The approximate concentrations of gallic acid in each of
the calibration standard solutions are presented in Table 2017.13.
(b)  Preparation of sample test solution.—Accurately
weigh about 100 mg test material and transfer into a 100 mL
volumetric flask. Add about 75 mL water and sonicate in an
ultrasonic bath containing about 3 cm water for up to 10 min
until the solids are dissolved. Dilute to volume with water and
mix well. Quantitatively pipette 5.0 mL of this solution into a
100 mL volumetric flask. Dilute to volume with water and mix
well. The weight of test material can be adjusted based on total
phenolic content in order to achieve an absorbance within the
calibration curve range.
where M = mass of gallic acid (g); P = purity of gallic acid
(%); and W = water content of gallic acid (expressed as decimal,
determined by Karl Fischer method or Loss on Drying method).
(b)  Calibration standard solutions.—Using the concentration
of gallic acid of the stock standard solution, calculate the actual
concentration of each calibration standard solution as follows:
s×v
Calibration standard solution (mg/L) =
25 mL
where S = the concentration of the stock standard solution
(mg/L) and V = the volume of stock standard solution (mL).
(c)  Calibration curve.—Plot absorbance at 765 nm vs
concentration of the calibration standards. Using linear
regression (forced through zero), calculate the slope, y-intercept,
and r2 value of the calibration curve for gallic acid.
(d)  System suitability.—The r2 value of the calibration curve
must be ≥0.990.
(e)  Total phenolic content in test material as is.—Calculate
the total phenolic content of the test material, in % w/w as is,
as follows:
A−b V ×D
Total phenols (% w/w, as is) = × × 100
m W × 1000
where A = the absorbance of the sample test solution at
765 nm; b = the y-intercept of the calibration curve; m = the
slope of the calibration curve; W = the weight of the test material
(mg); V = the volume of the sample test solution (100 mL);
1468  Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018

D = the dilution factor (20); and 1000 = the conversion from mL
to L. Note that values are expressed as gallic acid equivalents.
(f)  Total phenolic content in test material on dry weight
basis.—Calculate the total phenolic content of the test material,
in % w/w on a dry weight basis, as follows:
Total phenols as is
Total phenols (% w/w, dry) =
M
where M = the dry weight fraction of the test material,
expressed as (100 – % moisture)/100. The % moisture of the
test material can be determined by standard methods such as the
Karl-Fischer vacuum oven or the Halogen Moisture Analyzer
(Mettler Toledo HR73) methods. Note that values are expressed
as gallic acid equivalents.
Recovery
Maltodextrin was used as a representative carrier for extracts
in two recovery experiments. In the first, maltodextrin was
spiked with gallic acid at three levels totaling 400 mg: 70%
(w/w; 120 mg maltodextrin + 280 mg gallic acid), 30% (w/w;
280 mg maltodextrin + 120 mg gallic acid), and 5% (w/w;
380 mg maltodextrin + 20 mg gallic acid). Each level (400 mg)
was prepared in triplicate, extracted with 100 mL water, and
analyzed by the method on 3 separate days. In the second
experiment, 100 mg maltodextrin was mixed with either 100,
50, or 10 mg grape seed extract in triplicate, extracted with 100
mL water, and analyzed on 3 separate days.

Single-Laboratory Validation Selectivity

The single-laboratory validation (SLV) was performed at Nonphenolic compounds typically found in extracts were
Constellation Wines U.S. Mission Bell Winery in Madera, CA. tested for positive interference (cross-reactivity) in the method.
Compounds tested included glucose, fructose, and sucrose at
Test Materials 1 mg/mL.

A single lot of each test material was used throughout the Ruggedness
validation study.
(a)  Grape seed extract.—Dried extract supplied by
A ruggedness study was conducted in which seven factors
Polyphenolics, a division of Constellation Brands (Madera,
were varied in eight combinations according to the Youden
CA). Batch 09272503-01. Stored at 2–8°C protected from light.
and Steiner method (5). The method factors varied included the
(b)  Grape skin (pomace) extract.—Dried extract supplied by
heating apparatus, test material weight, read time after reaction,
Polyphenolics. Batch 12972503-11. Stored at 2–8°C protected
amount of Folin-C reagent, reaction time, reaction temperature, and
from light.
amount of Na2CO3. The high and low values for each factor are
(c)  Green coffee extract.—Dried extract supplied by Applied
listed in Table 1, and the factor combinations are listed in Table 2.
Food Sciences (Austin, TX). Standardized to 50% chlorogenic
acid and 65% total polyphenols on a dry weight basis. Batch Table 1.  Method factors and values for ruggedness testing
GCA A000912. Stored at 2–8°C protected from light.
(d)  Black tea extract.—Dried extract supplied by Applied Method factor High value Low value
Food Sciences. Standardized to 25% theaflavins and 50% Heating apparatus A = Heating block a
a = Water bath
catechins. Batch AFS 040201-A. Stored at 2–8°C protected a
Test material B = 200 mg extract b = 100 mg extract
from light. weight
(e)  Cocoa extract.—Dried extract supplied by The Hershey
Read time after C = 15 min c = 0 mina
Company (Hershey, PA). Stored at 2–8°C protected from reaction
light.
Amount of Folin-C D = 2 mL d = 1 mLa
(f)  Maltodextrin.—Archer Daniels Midland, Clintose-CR10 reagent
(10D.E. Maltodextrin). Used as negative control.
Reaction time E = 120 mina e = 90 min
a
Linearity Reaction F = 40°C f = 30°C
temperature

A calibration curve was generated in duplicate on each of Amount of Na2CO3 G = 3.0 mLa g = 2.0 mL
4 days. Each set of calibration standard data was plotted and a
  Indicates actual method value.
analyzed by linear regression. Residuals were calculated and
plotted to look for nonrandom patterns as a test for linearity. Table 2. Youden factor combinations for ruggedness
testing
Repeatability Precision
Measurement
Combination Factor values obtained
Four replicate preparations of each test material were made on
1 ABCDEFG X1
each of 4 separate days for a total of 16 analyses for each material.
For grape seed extract and grape skin extract, 200 mg test 2 ABcDefg X2
portions were extracted in 100 mL water; for black tea extract, 3 AbCdEfg X3
400 mg test portions were extracted in 200 mL water; for green 4 AbcdeFG X4
coffee extract, 400 mg test portions were extracted in 100 mL
5 aBCdeFg X5
water; and for cocoa extract, 3 g test portions were extracted in
6 aBcdEfG X6
200 mL water. Black tea and cocoa required extended sonication
for 45 min. Total phenolic content was determined as well as 7 abCDefG X7
within-day, between-day, and total RSDr for each material. 8 abcDEFg X8
 Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018  1469

Each factor value is represented four times in the eight
combinations. Grape seed extract was used as the test material
and analyzed once for each combination.
Results and Discussion
Linearity
Linearity was evaluated in duplicate on each of 4 days
using the calibration standard solutions. Plots are presented in
Figure 1, and a summary of the linear regression analyses is
presented in Table 3. The slopes were very consistent across
all of the runs, and the correlation coefficients varied from
0.996 to 1.000. To assess linearity, the residuals (expressed as
a percentage) were plotted as a function of concentration. The
second run on Day 1 appeared to show a concave pattern of
residuals, suggesting a nonrandom effect, but the other seven
runs showed random patterns of residuals, supporting a linear
relationship. The lowest concentration in the second run on
Day 1 had a residual of 16%, while all other residuals on that

(a) 1.4 (e) 1.4

y = 0.0054x + 0.0735 y = 0.0057x + 0.0194
Absorbance at 765 nm

1.2

Absorbance at 765 nm
1.2
R2= 0.9925 R2 = 0.9997
1 1
y = 0.0058x + 0.0503 y = 0.0059x + 0.0031
0.8 0.8
R2 = 0.9917 R² = 0.9986
0.6 0.6
0.4 0.4
0.2 0.2
0 0
0 50 100 150 200 250 0 50 100 150 200 250
Concentration, μg/mL Concentration, μg/mL

(b) 10.00% (f) 4.00%

5.00% 2.00%

0.00%
% Residual

0.00%
% Residual

0 50 100 150 200 250 0 50 100 150 200 250

−5.00% −2.00%

−10.00% −4.00%

−15.00% −6.00%

−20.00% −8.00%
Concentration, μg/mL Concentration, μg/mL

(c) 1.4 (g) 1.2

y = 0.0059x − 0.0037 y = 0.0054x + 0.0405
Absorbance at 765 nm

1.2 1
Absorbance at 765 nm

R² = 0.9998 R² = 0.9943
1
y = 0.0059x + 0.0062 0.8
0.8 y = 0.0056x + 0.0197
R2 = 0.999 0.6
0.6
R2 = 0.9991
0.4 0.4
0.2
0.2
0
0 50 100 150 200 250 0
Concentration, μg/mL 0 50 100 150 200 250
Concentration, μg/mL

(d) 3.00% (h) 8.00%

2.00% 6.00%
1.00% 4.00%
% Residual

2.00%
% Residual

0.00%
−1.00% 0 50 100 150 200 250 0.00%
−2.00% −2.00% 0 50 100 150 200 250

−3.00% −4.00%
−6.00%
−4.00%
−8.00%
−5.00%
Concentration, μg/mL Concentration, μg/mL

Figure 1.  Standard curve plots and residual plots from duplicate analyses on each of 4 days. (a) Standard curves on day 1; (b) residual
plots on day 1; (c) standard curves on day 2; (d) residual plots on day 2; (e) standard curves on day 3; (f) residual plots on day 3; (g) standard
curves on day 4; and (h) residual plots on day 4.
1470  Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018

Table 3.  Summary of linearity analyses RSDr values varied from 0.94 to 13.77%; and total RSDr
values varied from 4.81 to 15.18%. The highest RSDr values
Day Run Slope Y-intercept r2
corresponded to the extracts with the lowest phenolic content,
1 1 0.00545 0.0735 0.996 cocoa extract and green coffee extract. Grape seed extract,
2 0.00583 0.0503 0.996 grape skin extract, and black tea extract yielded RSDr values
2 1 0.00594 –0.0037 1.000
below 8% in all cases. Four of the five matrixes showed
higher between-day RSDr than within-day RSDr. While this
2 0.00587 0.0062 0.999
is not surprising, an F-test at the 95% confidence level was
3 1 0.00573 0.0194 1.000 conducted to see if the mean values were significantly different
2 0.00592 0.0031 0.999 between days. The analysis yielded significant P values for the
4 1 0.00543 0.0405 0.997 four matrixes displaying higher between-day than within-day
2 0.00560 0.0197 1.000 repeatability, indicating a significant difference between the
mean values for each day for those four matrixes.

day and subsequent days were 6% or less. Therefore, the data
overall demonstrate a linear relationship of absorbance to
concentration when simple linear regression is used to analyze
the calibration standards.
Repeatability Precision
Repeatability data are shown in Table 4. Grape seed extract
and grape skin extract had the highest total phenolic content
and cocoa extract had the lowest. Due to the low levels in cocoa
extract, a larger test portion was extracted so that absorbance
values were close to the lowest calibration standard. The within-
day RSDr values varied from 1.96 to 6.38%; between-day
Recovery
Recovery was determined by spiking either gallic acid
or grape seed extract into maltodextrin, a surrogate dietary
supplement carrier. The results of the spiking experiment using
gallic acid are shown in Table 5. At 70% gallic acid, the recovery
ranged from 94.8 to 100.4% over 3 days; at 30% gallic acid, the
recovery ranged from 93.7 to 95.3% over 3 days; and at 5%
gallic acid, the recovery ranged from 33.6 to 56.6% over 3 days.
The measured absorbance values for the 5% gallic acid spike
were far below the lowest calibration standard of the standard
curve and likely below the limit of quantitation of the method,
which would account for the low recovery values.

Table 4. Repeatability precision of extracts

Mean phenolic content, % w/w Mean

within-day Between-day F-test
Matrix N Day 1 Day 2 Day 3 Day 4 RSDr, % RSDr, % Total RSDr, % P value

Grape seed 4 74.55 81.53 79.20 76.85 7.47 0.94 7.53 0.401
extract
–6
Grape skin 4 73.58 81.43 83.83 72.48 2.62 7.14 7.60 6.6 × 10
extract
Black tea 4 51.35 55.43 57.23 54.68 1.96 4.39 4.81 4.6 × 10–5
extract
Green coffee 4 25.18 27.93 31.20 25.18 2.07 10.41 10.62 8.4 × 10–9
extract
Cocoa 4 2.00 2.40 2.65 2.00 6.38 13.77 15.18 6.4 × 10–5
extract

Table 5.  Spike recovery of gallic acid in maltodextrin

Absorbance
GA level, Content,
a
% Day Rep. 1 Rep. 2 Rep. 3 Mean % (w/w) Rec., %

70 1 0.763 0.767 0.776 0.769 66.4 94.8

2 0.830 0.854 0.808 0.831 70.3 100.4
3 0.744 0.756 0.756 0.752 66.7 95.3
30 1 0.322 0.338 0.335 0.332 28.1 93.7
2 0.347 0.316 0.363 0.342 28.1 93.7
3 0.298 0.306 0.310 0.305 27.3 91.0
5 1 0.036 0.038 0.038 0.037 2.25 45.0
2 0.036 0.034 0.039 0.036 1.68 33.6
3 0.027 0.027 0.027 0.027 2.83 56.6
a
  GA = Gallic acid.
 Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018  1471

Table 6.  Spike recovery of grape seed extract in maltodextrin

Absorbance
Content, Theoretical
GSEa, mg MDb, mg c
Day Rep. 1 Rep. 2 Rep. 3 Mean % (w/w) content, % (w/w) Rec., %

100 100 1 0.232 0.235 0.239 0.235 46.7 39.0 119.7

2 0.244 0.219 0.240 0.234 42.6 39.0 109.2
3 0.235 0.219 0.237 0.230 44.9 39.0 115.1
50 100 1 0.104 0.105 0.097 0.102 31.0 26.0 119.2
2 0.106 0.104 0.104 0.105 23.7 26.0 91.1
3 0.100 0.106 0.106 0.104 29.7 26.0 114.2
10 100 1 0.009 0.010 0.009 0.009 12.4 7.1 174.9
2 0.009 0.010 0.075 0.031 6.4 7.1 90.3
3 0.009 0.010 0.009 0.009 9.5 7.1 134.0
a
  GSE = Grape seed extract.
b
  MD = Maltodextrin.
c
  Based on GSE mean phenolic content of 78.03% from Table 4 and dilution into MD.

The grape seed extract spiking experiment is shown in
Table 6. The phenolic content was compared with the calculated
phenolic content of the spiked sample based on the mean
phenolic content of the grape seed extract from Table 4 (mean
over 4 days = 78.03%) and the proportions of grape seed extract
and maltodextrin in the samples. In this case, the recoveries
generally exceeded 100%, varying from 90.3 to 175%. The raw
absorbance values for the 50 and 10 mg spikes, however, were
below the value of the lowest calibration standard and likely
below the limit of quantitation of the method. Therefore, if we
only consider the 100 mg spiked samples, the recovery ranges
from 109 to 120%.
Selectivity
Glucose, fructose, and sucrose were examined for positive
interference in the method. Positive interference is defined as
a nonphenolic compound resulting in a positive response by
the method. Solutions of each sugar at 1 mg/mL were analyzed
by the method and all yielded absorbance values of 0.00,
demonstrating no positive interference.
Ruggedness
The results of the ruggedness trial are presented in Table 7.
For each combination of factor values, an absorbance value
was obtained and was converted to % total phenolic content.
To pull out the effect due to each factor, measurements
X1–X8 were analyzed to find the difference of means between
the high and low values of each factor. For example, the effect
of changing from a heating block to a water bath was assessed
by taking the mean result of combinations 1–4, which used the
heating block, and subtracting the mean result of combinations
5–8, which used the water bath. These differences of means
are presented in Table 8. When the effects are calculated based
on raw absorbance values, there appears to be a significant
difference between factor values B and b. Because this method
factor is test portion weight (200 mg versus 100 mg), it
makes sense that there is a difference in the raw absorbance
values. When the test portion size is taken into account in the
calculations of % total phenolic content and Δ % total phenolic
content, there is no longer a significant difference observed. All
factor effects showed an absolute Δ % total phenolic content
of 7.3 or less, indicating that none of the factor values cause a
significant difference in the method performance.
Comparison of Method Performance to SMPR®
2015.009
In 2015, the AOAC Stakeholder Panel for Dietary
Supplements approved Standard Method Performance
Requirements (SMPRs) for the estimation of total phenolic

Table 7. Results of ruggedness trial

Measurement
Combination Factor values obtained Abs value Content, %

1 ABCDEFG X1 0.489 81.95

2 ABcDefg X2 0.513 86.14
3 AbCdEfg X3 0.288 93.75
4 AbcdeFG X4 0.257 82.93
5 aBCdeFg X5 0.531 89.28
6 aBcdEfG X6 0.496 83.18
7 abCDefG X7 0.231 73.86
8 abcDEFg X8 0.254 81.88
1472  Kupina et al.: Journal of AOAC International Vol. 101, No. 5, 2018

Table 8.  Effect of factor values in ruggedness trial

Factor effect Calculation Effect Δ Abs value Δ Content, %

A and a [(X1 + X2 + X3 + X4)/4] – [(X5 + X6 + X7 + X8)/4] J 0.009 6.19

B and b [(X1 + X2 + X5 + X6)/4] – [(X3 + X4 + X7 + X8)/4] K 0.249 3.04
C and c [(X1 + X3 + X5 + X7)/4] – [(X2 + X4 + X6 + X8)/4] L 0.005 1.18
D and d [(X1 + X2 + X7 + X8)/4] – [(X3 + X4 + X5 + X6)/4] M –0.021 –6.32
E and e [(X1 + X3 + X6 + X8)/4] –[(X2 + X4 + X5 + X7)/4] N –0.001 2.14
F and f [(X1 + X4 + X5 + X8)/4] –[(X2 + X3 + X6 + X7)/4] O 0.001 –3.75
G and g [(X1 + X4 + X6 + X7)/4] –[(X2 + X3 + X5 + X8)/4] P –0.029 –7.28

Table 9.  Comparison of SMPR 2015.009 and the Folin-C the five matrixes showed >7% RSDr. No data were collected
SLV results for levels less than 2% (w/w) total phenolic content, and the
method has a range of about 5–100% total phenolic content,
SMPR 2015.009
higher than the range specified in SMPR 2015.009.
SLV (gallic acid
parameter equivalents) SLV result
a
Conclusions
Analytical 5–500 mg/L 40–200 mg/L for GA
b
range 6–100% (w/w) for GSE
The Folin-C method meets the recovery and repeatability
LOQ ≤5 mg/L 40 mg/L for GA
requirements for the levels tested and was granted First Action
6% (w/w) for GSE
status Official Method 2017.13.
Recovery 80–110% 91–104% for GA
RSDr 3–5 mg/L ≤9% (w/w) NDc
References
>5 mg/L ≤7% (w/w) <7.5% (w/w)
a
  GA = Gallic acid. (1) Pandey, K.B., & Rizvi, S.I. (2009) Oxid. Med. Cell. Longev.
b 2, 270–278. doi:10.4161/oxim.2.5.9498
  GSE = Grape seed extract.
(2) Graf, B.A., Milbury, P.E., & Blumberg, J.B. (2005) J. Med.
c
  ND = Not determined. Food 8, 281–290. doi:10.1089/jmf.2005.8.281
(3) Grases, F., Prieto, R.M., Fernández-Cabot, R.A., Costa-Bauzá, A.,
Sánchez, A.M., & Prodanov, M. (2015) Nutr. J. 14, 94.
content using the Folin-C assay (6). Table 9 compares the
doi:10.1186/s12937-015-0083-3
method performance requirements from SMPR 2015.009 to (4) Singleton, V.L., & Rossi, J.L. (1965) Am. J. Enol. Vitic. 16, 144–158
the results of the SLV described here. The recovery for (5) Statistical Manual of the AOAC (1975) AOAC
maltodextrin spiked with gallic acid meets the requirement of INTERNATIONAL, Gaithersburg, MD
80–110%. The within-day RSDr at >5 ppm is required to be (6) Official Methods of Analysis (2015) 20th Ed., AOAC
≤7% and in the SLV was observed to be <7.5%. Only one of INTERNATIONAL, Rockville, MD, AOAC SMPR 2015.009