ANALYTICAL SCIENCES DECEMBER 2012, VOL.

28 1179
2012 © The Japan Society for Analytical Chemistry

Evaluation of a Method to Quantify Quercetin Aglycone in Onion

(Allium cepa) by Single- and Multi-laboratory Validation Studies
Jun WATANABE,*† Jun TAKEBAYASHI,** Yuko Takano-ISHIKAWA,* and Akemi YASUI*

*National Food Research Institute, National Agriculture and Food Research Organization,
2-1-12 Kannondai, Tsukuba, Ibaraki 305–8642, Japan
**National Institute of Health and Nutrition, 1-23-1 Toyama, Shinjuku, Tokyo 162–8636, Japan

Official Methods of Analysis of AOAC International (OMA) 2006.07 was originally designed for quantifying flavonol
aglycones in ginkgo dietary supplements. To determine whether the method is applicable to the quantification of flavonol
aglycones in lyophilized onion samples, single- and multi-laboratory validation studies were performed. Triplicated
measurements on 3 different days revealed that the mean quercetin content was 3.48 g/kg dry weight, and the relative
repeatability standard deviation (RSDr) and the relative intermediate standard deviation (RSDint) were 0.8 and 1.8%,
respectively. The recovery of quercetin-3-O-glucoside spiked at 3 different amounts (1.56, 3.12, and 6.24 g/kg dry weight
of onion) ranged from 98.42 to 100.31%, and the RSDr and RSDint ranged from 2.2 to 5.9%, and from 3.4 to 5.2%,
respectively. A multi-laboratory validation study showed that the mean quercetin contents were 2.80 and 6.61 g/kg dry
weight, and that satisfactory inter-laboratory precision (RSDr and RSDR ranged from 0.41 to 0.92%, and from 6.73 to
7.62%, respectively); all HorRat values were less than 2. These results indicate that OMA 2006.07 is applicable to the
determination of the quercetin content of lyophilized onion samples.

(Received July 30, 2012; Accepted November 2, 2012; Published December 10, 2012)

quantification of quercetin in lyophilized onion samples. Single-

Introduction
Flavonoids are a group of polyphenol secondary metabolites
that play an important role in the normal growth, development
and defense of plants.1 Onions are a rich source of flavonoids2
and one of the main sources of polyphenols in the human diet.3
The main flavonoids in onions are quercetin mono- and
di-glucosides, which account for up to 80% of the total content
of flavonoids.1,4 There is increasing interest in quercetin and
related compounds due to their possible health benefits,
including inhibiting the growth of several kinds of cancer cells5–7
as well as their antioxidant and free-radical scavenger
activities.8,9
The content of polyphenols varies depending on the variety of
onion,10 the environmental and growth conditions, the time of
harvest, and the processing and storage conditions.11 Thus,
using a validated method to quantify the quercetin content of
onions would aid in the selection of onion cultivars with higher
quercetin content, and help to increase quercetin content by
cultivation management. A validated method for the
quantification of flavanol aglycones, including quercetin, is
provided in Official Methods of Analysis of AOAC International
(OMA) 2006.07 Flavonol Aglycones in Ginkgo biloba Dietary
Supplement Crude Materials and Finished Products, but this
method is applied to ginkgo products.12 In this method, the
flavanol content is determined by HPLC after all of the flavonol
glycosides are hydrolyzed into aglycones. We aimed to
determine whether OMA 2006.07 is applicable to the
†
To whom correspondence should be addressed.
E-mail: nabej@affrc.go.jp
and multi-laboratory validation studies were performed to
determine the intra- and inter-laboratory precisions, respectively.
Quercetin 3-O-glucoside was spiked into onion samples, and its
recovery was quantified to define the trueness of the method.
Experimental
Chemicals
Quercetin dihydrate was purchased from Wako Pure Chemical
Ind. (Osaka, Japan) and used as a standard. Quercetin
3-O-glucoside was purchased from Extrasynthese (Lyon,
France) and purified by immobilizing on a solid-phase extraction
cartridge (Oasis HLB; Waters Japan Tokyo, Japan) and eluting
with methanol. The purity of the purified quercetin
3-O-glucoside was more than 99% based on HPLC analysis.
Other chemicals were of reagent grade.
Quantification of the quercetin content of onion samples
Onion samples were extracted in accordance with a method
for crude material (dried leaves of Ginkgo biloba) in OMA
2006.07,12 and the quercetin contents of the extracts were
analyzed according to OMA 2006.07.12 Briefly, about 200 mg
of lyophilized and pulverized onion powder was weighed into a
glass serum vial (30 mL), and a diluent (ethanol:water:HCl =
50:20:8 (v/v/v), 12 mL) was added. After mixing by sonication,
hydrolyzation and extraction were performed by heating at
90 ± 2° C for 60 ± 3 min in a vial capped with a Teflon-faced
septum. After cooling to ambient temperature, the volume of
the extract was adjusted to 25 mL with methanol, and then
filtered through a 0.45-μm PVDF membrane. Quercetin in the
1180
Fig. 1 Typical chromatogram of onion extract. A lyophilized and
pulverized onion sample was extracted and hydrolyzed, and the extract
was analyzed by HPLC according to OMA 2006.07.
extract was separated and quantified by HPLC using a
Phenomenex Prodigy ODS(3) column (4.6 × 250 mm;
Phenomenex, Torrance, CA) eluted with methanol:0.85 (v/v) %
phosphoric acid = 1:1 (v/v) at a flow rate of 1 mL/min. A
typical chromatogram for onion extract is shown in Fig. 1.
Single-laboratory validation
Onions (“Kitamomiji 2000”) were cultivated at Kitami
Agricultural Experimental Station, Hokkaido Research
Organization, in 2010. Non-edible parts were removed from the
bulbs, and about 1 kg of cleaned onion was snap-frozen with
liquid nitrogen. The frozen sample was lyophilized, then
pulverized in a grinder mill (GM-200; Retsch, Haan, Germany).
A methanol solution of quercetin-3-O-glucoside was added to
aliquots (about 200 mg each) of onion sample at doses of 0,
1.56, 3.12, and 6.24 g/kg, and then the mixtures were incubated
at ambient temperature for 1 h to evaporate the methanol.
Spiked and non-spiked samples were hydrolyzed as described
above; then, the quercetin concentration and recovery were
calculated. Triplicate measurements were repeated on 3
different days.
Multi-laboratory validation
The onions were as described above. Bulbs without the
brownish outer skin (sample A) or with small amounts of outer
skin (sample B) (about 5 kg each) were snap-frozen with liquid
nitrogen. Frozen samples were lyophilized, and then pulverized
in a grinder mill (GM-200). Aliquots (about 300 mg) of each
onion sample were portioned into 20 glass vials; 10 vials were
used for the homogeneity test, and the remaining vials were
stored at –30° C until transport to the validation participants.
Upon receipt of the test samples, participants were asked to
immediately store them in a refrigerator (4° C) until use.
The multi-laboratory validation study was performed with 4
participating laboratories and 2 kinds of test materials (A and B,
described above). Participants were raised from the laboratories
joining the grant-in-aid for the research project shown in the
Acknowledgments section. Each sample was provided as
randomly labeled on, blind duplicates, and each contained a test
portion. Participants were also provided with a method protocol
as well as an electronic evaluation and reporting sheet (MS
Excel format). Four test samples were analyzed once under
repeatability conditions.
Statistical analyses
The repeatability, intermediate and reproducibility standard
deviations for the quercetin content and recovery were calculated
by one-way analysis of variance.
ANALYTICAL SCIENCES DECEMBER 2012, VOL. 28
Table 1 Repeated measurements for the determination of the
quercetin content of onion samples on dry basis
Quercetin content/g kg–1
Day 1 Day 2 Day 3
1 3.40 3.54 3.54
2 3.45 3.51 3.47
3 3.39 3.51 3.49
Mean quercetin content/g kg–1 3.48
Sr/g kg–1 a 0.03
RSDr, %b 0.8
Sint/g kg–1 c 0.06
RSDint, %d 1.8
a. Sr = Repeatability standard deviation.
b. RSDr = Repeatability relative standard deviation.
c. Sint = Intermediate precision standard deviation.
d. RSDint = Intermediate precision relative standard deviation.
Results and Discussion
Precision within a laboratory
The quercetin content of onion samples was measured in
triplicate, and the measurements were repeated on 3 different
days. The mean quercetin content was 3.48 g/kg dry weight,
and the relative repeatability standard deviation (RSDr) and the
relative intermediate standard deviation (RSDint) were 0.8 and
1.8%, respectively (Table 1). The value of RSDr was compared
with the predicted levels of precision obtained from the Horwitz
equation,
Predicted RSDr = C–0.15,
where C is the commonly measured concentration of the analyte
in the sample, expressed as a mass fraction. The mass fraction
of the mean quercetin content was calculated by multiplying the
volume concentration by the molecular weight of quercetin
dihydrate. The predicted RSDr was calculated to be 2.34%,
which is larger than the values of RSDr and RSDint. Thus, both
the repeatability and the intermediate precision within a
laboratory for the quantification of quercetin content of onion
samples were judged to be good.
Spike and recovery tests
In OMA 2006.07, the flavanol content, based on the aglycone
concentration, is determined by HPLC following acid hydrolysis.
Complete hydrolysis of quercetin glucosides in onions is
required to obtain values with satisfactory trueness. The
dominant quercetin derivatives in onions are reported to be
quercetin 3,4′-O-diglucoside and quercetin 4′-O-glucoside.4
In  our preliminary experiments, 4′-O-glucoside was more
susceptible to acid hydrolysis than 3-O-glucoside (data not
shown). Thus, we chose quercetin-3-O-glucoside, which is
more resistant to acid hydrolysis, for spike and recovery tests.
Quercetin-3-O-glucoside was added to onion samples, and the
recoveries were determined. Since onion samples that contained
no quercetin were not available, the quercetin contents of onion
samples without adding quercetin-3-O-glucoside were measured
under repeatability conditions, and the average quercetin content
(3.48 g/kg dry weight) was subtracted from the quercetin
contents of spiked samples. The spiked amounts of
quercetin-3-O-glucoside (1.56, 3.12, and 6.24 g/kg dry weight
ANALYTICAL SCIENCES DECEMBER 2012, VOL. 28 1181

Table 2 Results of spike and recovery tests of quercetin-3-O-glucoside in onion samples on dry basis

Recovery, %
Spiked amount/
1.56 3.12 6.24
g kg–1
Day 1 Day 2 Day 3 Day 1 Day 2 Day 3 Day 1 Day 2 Day 3

1 106.20 104.93 100.19 96.33 104.86 98.82 99.27 101.74 98.23

2 100.41 101.41 98.00 97.85 102.74 97.96 96.91 101.80 98.73
3 87.48 99.27 101.66 103.33 102.98 97.89 90.70 101.72 96.73
Mean recovery, % 99.95 100.31 98.42
Sr, %a 5.87 2.25 2.67
RSDr, %b 5.9 2.2 2.7
Sint, %c 5.16 3.37 3.77
RSDint, %d 5.2 3.4 3.8

a. Sr = Repeatability standard deviation. b. RSDr = Repeatability relative standard deviation. c. Sint = Intermediate precision standard
deviation. d. RSDint = Intermediate precision relative standard deviation.

Table 3 Homogeneity test results of 2 kinds of onion samples Table 4 Multi-laboratory validation study results for the
for the multi-laboratory validation study determination of quercetin content of onion samples on dry basis

Quercetin content/g kg–1a Quercetin content/g kg–1

Sample A Sample B Sample A Sample B

1 2 1 2 1 2 1 2

1 2.96 2.95 6.58 6.69 A 2.60 2.61 6.44 6.36

2 2.93 2.97 6.51 6.73 B 2.61 2.62 6.17 6.22
3 3.00 2.95 6.68 6.84 C 3.01 2.99 7.17 7.26
4 2.92 2.96 6.74 6.59 D 2.96 2.95 6.58 6.69
5 2.94 2.88 6.70 6.57 Number of laboratories 4 4
6 2.91 2.90 6.76 6.70 Mean/g kg–1 2.80 6.61
7 2.87 2.86 6.48 6.55 Sr/g kg–1 a 0.01 0.06
8 2.88 3.04 6.69 6.66 RSDr, %b 0.41 0.92
9 2.88 2.84 6.64 6.64 SR/g kg–1 c 0.21 0.44
10 2.88 2.85 6.76 6.62 RSDR, %d 7.62 6.73
Mean/g kg–1a 2.92 6.66 PRSDR, %e 4.85 4.26
Fb 2.02 1.17 HorRatf 1.57 1.58

a. Quercetin contents of onion samples were shown on dry basis.
b. F = F value of one-way ANOVA. When F value was less than F
critical value (4.04, α = 0.05), the sample judged to be homogeneous.
of onion) were approximately comparable to 25, 50 and 100%
of the quercetin content in the onion bulbs (Table 1), the average
recoveries were 99.95, 100.31, and 98.42%, and RSDr and
RSDint were 5.9, 2.2, and 2.7%, and 5.2, 3.4, and 3.8%,
respectively (Table 2). According to the single-laboratory
validation guidelines of AOAC,13 the recovery limit of spiked
analyte is 92 – 105%. These results clearly indicate that the
trueness of the method for quantifying the quercetin content of
onion samples can be judged to be good.
Multi-laboratory validation
Homogeneity test: Two kinds of onion samples containing
different amounts of quercetin were prepared, and the
homogeneity of each sample was verified. From each test
material, 10 samples (units) were taken at random from the
filled vials, and each sample was split into 2 equal parts (unit
subsamples). The quercetin content of each unit subsample was
measured under repeatability conditions, i.e., the same method
was conducted on identical test items in the same laboratory by
the same operator using the same equipment within a short
period of time. The within- and between-unit standard
a. Sr = Repeatability standard deviation.
b. RSDr = Repeatability relative standard deviation.
c. SR = Reproducibility standard deviation.
d. RSDR = Reproducibility relative standard deviation.
e. PRSDR = predicted RSDR (2C–0.1505; C, mass fraction).
f. HorRat = RSDR/predicted RSDR.
deviations for quercetin content determinations were calculated
by one-way analysis of variance, and by applying the F-test at
the 95% confidence level. All of the p values of the test
materials for the validation study were larger than 0.05,
indicating that the samples were homogeneous (Table 3).
Inter-laboratory precision: Individual values obtained at the 4
participating laboratories are given in Table 4. The mean
quercetin contents were 2.80 and 6.61 g/kg dry weight. The
RSDr and reproducibility relative standard deviation (RSDR) of
the quercetin content ranged from 0.41 to 0.92%, and from 6.73
to 7.62%, respectively (Table 4). The precision data obtained in
the inter-laboratory study were compared with the predicted
levels of precision obtained from the Horwitz equation,
Predicted RSDR = 2C–0.15,
where C is the commonly measured concentration of the analyte
in the sample, expressed as the mass fraction. The mass
1182
fractions of the mean quercetin content of samples A and B
were calculated in the same way as the predicted RSDr, and
provided values of 4.85 and 4.26%, respectively, according to
the Horwitz equation. The HorRat value, which is the ratio of
RSDR (measured) to the predicted RSDR (Horwitz), gives a
comparison between the actual precision and the precision
predicted by the Horwitz equation. The calculated HorRat value
can be used as a performance parameter to indicate the
acceptability of the precision of the method. A HorRat value of
<2 usually indicates satisfactory reproducibility, whereas a value
of >2 usually indicates unsatisfactory performance of the
method. The HorRat values for the quercetin content in samples
A and B were 1.57 and 1.58, respectively (Table 4). All HorRat
values for the onion samples were lower than 2, thus indicating
that this method based on OMA 2006.07 can be used to quantify
the quercetin content of onion samples with satisfactory
inter-laboratory precision.
Acknowledgements
This work was supported by a grant-in-aid for research projects
(Development of Fundamental Technology for Analysis and
Evaluation of Functional Agricultural Products and Functional
Foods) from the Ministry of Agriculture, Forestry and Fisheries
of Japan. Onion samples were kindly provided by Dr. Daisuke
Yanagida of Kitami Agricultural Experimental Station, Hokkaido
Research Organization. We express our appreciation to Dr.
Takashi Yamagishi (Kitami Institute of Technology) and Dr.
Takato Muro (National Agriculture and Food Research
Organization, Hokkaido Agricultural Research Institute) for
their participation in this study.
ANALYTICAL SCIENCES DECEMBER 2012, VOL. 28
References
1. S. Sellappan and C. C. Akoh, J. Agric. Food Chem., 2002,
50, 5338.
2. K. H. Miean and S. Mohamed, J. Agric. Food Chem., 2001,
49, 3106.
3. P. Brat, S. George, A. Bellamy, L. Du Chaffaut, A. Scalbert,
L. Mennen, N. Arnault, and M. J. Amiot, J. Nutr., 2006,
136, 2368.
4. P. Bonaccorsi, C. Caristi, C. Gargiulli, and U. Leuzzi, J.
Agric. Food Chem., 2005, 53, 2733.
5. N. Hosokawa, Y. Hosokawa, T. Sakai, M. Yoshida, N.
Marui, H. Nishino, K. Kawai, and A. Aoike, Int. J. Cancer,
1990, 45, 1119.
6. M. G. Hertog, P. C. Hollman, M. B. Katan, and D.
Kromhout, Nutr. Cancer, 1993, 20, 21.
7. P. Aron and J. Kennedy, Mol. Nutr. Food Res., 2008, 52, 79.
8. K. Murota and J. Terao, Arch. Biochem. Biophys., 2003,
417, 12.
9. P. M. Clifton, J. Biomed. Biotechnol., 2004, 2004, 272.
10. J. Yang, K. J. Meyers, J. van der Heide, and R. H. Liu, J.
Agric. Food Chem., 2004, 52, 6787.
11. C. Manach, A. Scalbert, C. Morand, C. Remesy, and L.
Jimenez, Am. J. Clin. Nutr., 2004, 79, 727.
12. AOAC Official Methods of Analysis, 18th ed., 2010, AOAC
International, Gaithersburg, MD, Method 2006.07.
13. AOAC guidelines for single laboratory validation of
chemical methods for dietary supplements and botanicals,
http://www.aoac.org/Official_Methods/slv_guidelines.pdf.