Food Analytical Methods

https://doi.org/10.1007/s12161-018-1337-4

Extraction, Purification, and Determination by HPLC

of Quercetin in Some Italian Wines
F. Buiarelli 1 & F. Bernardini 2 & P. Di Filippo 3 & C. Riccardi 3 & D. Pomata 3 & G. Simonetti 1 & R. Risoluti 1

Received: 15 May 2018 / Accepted: 29 July 2018

# Springer Science+Business Media, LLC, part of Springer Nature 2018

Abstract
In this paper, we report a simple method for the determination of quercetin, a typical antioxidant compound ubiquitously present
in vegetable food products including grapes and wine. To this aim, 12 Italian wines were analyzed and the amount of quercetin
was determined by high-performance liquid chromatography with ultraviolet detector. To reduce the interferences, a minimal
sample preparation was performed on solid phase cartridge, then the samples were eluted through a narrow bore C18 column
under gradient elution in less than 25 min. Nine wines were red and three white. The amount of quercetin ranged from 0.5 to
4.3 mg L−1. The method was evaluated in terms of reproducibility, linearity, recovery, and limits of detection and quantification,
and it resulted relatively fast, economical, and suitable for those laboratories involved in routine analysis. In addition, the
possibility to determine the content of quercetin derivates, as free quercetin, after acidic hydrolysis was investigated. Despite
the limited number of analyzed samples, the results from this explorative study are in agreement to those from other surveys.
Moreover, due to the problems arising from hydrolysis, further research are needed for determining each single quercetin derivate
in wine, in order to better characterize its active compounds.

Keywords Quercetin . High-performance liquid chromatography . Wine . UV detection . Quantitative analysis

Introduction the consumption of red wine, rich in antioxidant substances

Quercetin, a flavonol natural antioxidant, is contained in sev-
eral foods and beverages including wines (David et al. 2016;
Garrido and Borges 2013). Several studies have demonstrated
its potential chemopreventive activity and its anti-inflamma-
tory, anti-allergic, anti-bacteric, and vasodilatory properties
(Ang et al. 2014). In addition to food, there are many supple-
ments containing quercetin, available on the market, declaring
beneficial effects, but the results are still controversial
(Mohammadi-Sartang et al. 2017; Sahebkar 2017).
Starting from the early 1990s, a lot of studies tried to ex-
plain the French paradox (Ferrières 2004) as closely linked to
* F. Buiarelli
francesca.buiarelli@uniroma1.it
1
Department of Chemistry, University BLa Sapienza^, P.le Aldo Moro
5, 00185 Rome, Italy
2
Economics and Business Administration University BRoma Tre^,
Via Silvio d’Amico 77, 00145 Rome, Italy
3
Inail DIT, Via Roberto Ferruzzi, 00143 Rome, Italy
including quercetin (Renaud and De Lorgeril 1992;
McDonald et al. 1998). At the moment, there are no still de-
finitive conclusions, but anyway it is universally assumed that
drinking a glass of wine a day and eating more fruits and
vegetables, since the right balance of diet is more important
than any single component, could be helpful in preventing
disease (Vendrame 2013; Lante et al. 2004).
Quercetin can occur in food as aglycone (known also as
3,5,7,3′,4′-pentahydroxyflavone) or in a bonded form (quer-
cetin-O-glucosides or glucuronides) (Materska 2008).
Analysis of quercetin is usually carried out by different tech-
niques in different matrices such as food (Tokusoglu et al. 2003;
Wach et al. 2007), plasma (Abdelkawy et al. 2017), and pollen
(Chen et al. 2015).
As for wines, one of the most consumed products in the
world, several authors since the late 1990s (McDonald et al.
1998) have already focused on quercetin in wines. More re-
cently in 2012, Belajova (2012) proposed some phenolic con-
stituents, including quercetin, as possible markers of wines.
In order to better characterize Italian wines, in line with our
previous works (Buiarelli et al. 2010, 2007), for increasing the
information about the simultaneous presence of active

compounds in wine, we optimized a relatively rapid and reli-
able method for the analysis, after a step of purification, and for
the determination of the content of quercetin by high-
performance liquid chromatography (HPLC) coupled to an ul-
traviolet (UV) detector. The method was validated in terms of
recovery/accuracy, linearity, repeatability, limit of detection
(LOD), and limit of quantification (LOQ). Finally, it was ap-
plied to different Italian wines (red and white) and the results
were compared to those obtained, in the same or in other coun-
tries, by other authors (Ragusa et al. 2017; Faustino et al. 2003;
Careri et al. 2003; Lante et al. 2004; Gambelli and Santaroni
2004; Vuorinen et al. 2000).
In addition, in order to determine total quercetin glucosides/
glucuronides as free aglycone, acidic hydrolysis was carried out
on wine samples. The degradation of the formed quercetin in an
acid medium confirmed the complexity of determining the total
contribution of these active compounds.
Material and Methods
Chemicals, Reagents, and Materials
Ultrapure water was produced with a Pure Lab System (USF
Elga, Germany). Methanol Plus (HPLC grade), diethyl ether
anhydrous (> 99%), formic acid (91%), and quercetin
dihydrate were from Sigma-Aldrich (Milan, Italy).
Cartridges were from bakerbond spe C18 -500 mg, 3 mL
(JTBaker, Phillipsburg, NJ, USA). Sample filtration was car-
ried out by 0.45-μm, 4-mm internal diameter, Syringe-driven
Filter Units, Millex-HN (Millipore Corporation, Bedford,
MA, USA). All the other reagents were of analytical grade.
Stock standard solutions of 1 mg/mL in methanol, (1 mg of
the compound was weighed on Gibertini balance with
0.01 mg sensitivity) were prepared monthly and stored at −
20 °C in the dark. Working solutions were daily prepared in
methanol by appropriate dilution. A group of 12 commercially
available wines (vintage 2016) from different Italian regions
(from NI north of Italy, CI central Italy, and SI south Italy) was
analyzed. Nine of them were red and three white. They were
labeled as brands 1 to 12. Wine samples were opened,
protected against sunlight, and stored at 4 °C, except wine 9.
Analyses were carried out within few days. After sample prep-
aration, all the samples were filtered through a 0.45-μm mem-
brane filter before the chromatographic analysis.
Instrumentation
LC analysis was carried out by a Jasco PU 980 (Easton, MD,
USA) gradient HPLC system with two pumps with a high-
pressure mixing system. The injection valve was a Reodyne
7410 (Sigma-Aldrich—Milan, Italy) with a sample loop of
5 μL. The C18 column (L 250 mm × 2.1 mm ID) used was
Food Anal. Methods
slurry, packed in our laboratory according to a procedure pub-
lished by our laboratory (Benincasa et al. 1987), with
Nucleosil ODS 5 μm, obtained from Nacherey-Nagel D-
5160 Duren (Germany) and kept at room temperature during
the analysis. An ultra-low dead-volume precolumn filter
A.318 Upchurch Scientific (Oak Harbor WA 98277-9986)
was used. Detection was carried out at 260 nm by a rapid
scanning UV-vis detector Jasco 875-UV (Easton, MD, USA)
equipped with a 1.2-μL flow cell. Data were recorded by an
integrator, Hewlett Packard 3344A.
Mobile phase was MeOH-H 2 O at a flow rate of
150 μL min−1. The elution was carried out in gradient condition:
at time 0 min, MeOH is 40%, after 30 min, MeOH is 70%, and
then at 40 min, MeOH is 100%. Equilibration time was 10 min.
Quercetin was identified based on its retention time
(22.35 min) and UV spectrum, using an HPLC provided by
diode array of another laboratory (data not shown).
Sample Preparation
After evaporation of the alcohol, under vacuum below 30 °C,
4 mL of wine was loaded on cartridge C18, previously activat-
ed by MeOH (10 mL) and washed by water (4 mL). The
interferences were removed by 4 mL of acidic water (contain-
ing 1% of formic acid) and then the analyte was eluted by
6 mL of diethyl ether. After drying of the eluate under nitrogen
stream, the residue was dissolved in 100 μL of MeOH and
5 μL was injected, after sample filtration through a 0.45-μm
membrane, in the HPLC-UV system.
Method Validation
For the method validation, detection limit, quantification limit,
linearity, matrix effect, repeatability, and recovery/accuracy
were assessed. The calculation of limit of detection (LOD)
and limit of quantification (LOQ) was based on the analysis
of spiked blank of wine, a red wine free of quercetin, with the
analyte, before the whole procedure. The concentration of the
spiked sample producing a peak with a signal-to-noise (S/N)
ratio of 3/10 was chosen as LOD/LOQ. Two calibration curves
(A and B) were built in HPLC-UV. Curve BA^ was built to
evaluate the instrumental linearity, using five standard solutions
at increasing analyte concentrations (0.3–12 mg mL−1). Matrix-
matched calibration curve BB^ was prepared to evaluate the
method linearity and to estimate any possible matrix effect.
To mime the environment and the interactions between the
analyte and other compounds in the matrix, maybe altering
the analytical response, samples of blank of wine, free of quer-
cetin, were processed according to the analytical procedure in
sample preparation and spiked, prior to the injection with the
same standard solutions of curve BA.^ Matrix effect was deter-
mined by the ratio between the slope of curve (B) and the slope
of the standard calibration curve (A) (Buiarelli et al. 2015). The
Food Anal. Methods
repeatability was evaluated as intra-day (n = 10) and inter-day
repeatability within 5 days (n = 5), using control standard solu-
tion and fortified wines at the LOQ level and was expressed as
relative standard deviation (RSD%). The recovery rate, as a
measure of accuracy, was assessed comparing the results after
addition of a known amount of the compound (at LOQ level
and twice and three times more concentrated) to a blank wine
sample, before and after the cleanup.
Results and Discussion
Optimization of Sample Preparation and Recovery
The direct injection of wine after dilution resulted not effec-
tive, due to the high background noise, so we proceeded to a
simple and quick sample purification.
Preliminary experiments were done directly on 4 mL of a
standard solution of quercetin in water (at LOQ level) for the
choice of the conditions for the best extraction and/or purifica-
tion steps. We compared a direct extraction (by 6 mL of diethyl
ether) with the purification efficiency on a spe cartridge. Three
aliquots were tested with both the procedures for three times
giving a recovery > 90%. Successively, in order to verify the
cleanup procedure, 4 mL of a wine, free of quercetin (chosen as
blank), was spiked with the analyte at the three different levels.
The recovery was not dependent on the level of concentra-
tion and was calculated from the calibration curve (B).
Recovery calculation was R = C/Cref x × 100 where C is the
concentration found with the calibration curve and Cref is the
reference concentration (added before the procedure)
(Buiarelli et al. 2015). The simple extraction gave an average
recovery of 67% ± 4%, whereas the average recovery after spe
cartridge was higher than 87% ± 7%. We also tested MeOH as
eluting solvent from spe cartridge, but the main concern is due
to a higher extraction of anthocyanin, which causes interfer-
ences and higher background.
In addition, to investigate the possibility of measuring the
content of quercetin-O-glucosides, as free aglycone after acidic
hydrolysis, the stability of quercetin under acid medium was
tested. A standard solution of quercetin was added to 5 mL of
methanolic solution (50:50 v:v) of 1.2 M HCl and was kept for
2 h at 90 °C, in the presence of diethyldithiocarbamate as anti-
oxidant. The content of quercetin, analyzed before and after the
addition of the acidic solution, decreased to about 40%,
highlighting degradation of the compound in acidic medium.
The reduction to 1 h and to 80 °C did not improve the yield of
the process.
Matrix Effect, Linearity, LOD/LOQ, and Reproducibility
The matrix effect can greatly affect the reproducibility and
accuracy of the method. The linearity was checked for 3 days
by calibration curve both in solution and in matrix (adding the
analyte at the end of the cleanup). The linearity in the inves-
tigated ranges was very good, as demonstrated by the corre-
lation factors R2 ≥ 0.999. Matrix effects (ME), calculated ac-
cording to Buiarelli et al. (2015), was not negligible (> 10%);
therefore, curve (B) was used for the quantification of the
samples and the quantitative results were corrected by the
recovery.
Table 1 summarizes LOD and LOQ values for quercetin
expressed as mg L−1 and the other validation parameters for
quercetin by HPLC-UV.
Analysis of Real Samples
Italian wines are known worldwide for their broad variety and
for their taste. There are a lot of documented grape varieties in
circulation and usually many of Italy’s best red wines are
labeled with the name of the wine appellation, often in com-
bination with the grape variety.
Table 2 summarizes the quantitative results of quercetin,
found in 12 Italian wines, considering products of different
aging and vine. Despite the limited number of samples, it is
possible to argue some consideration, expressing assessments
with caution in order to avoid untimely conclusion.
In red wines (9 of 12), the quercetin value ranged between
0.5 and 4.3 mg L−1 in line to the values of other authors
(Vuorinen et al. 2000; McDonald et al. 1998), whereas in
white wines (3 of 12), it was < LOQ. Brands 3, 4, and 5,
although relative to different regions of Italy, are coming
from the same vine and the relative quercetin concentration
is quite similar. Brands 8 and 9 are the same wine, but the
second was stored not properly for a long time, and that means
quercetin undergoes degradation. These results are in
agreement to those found by Careri et al. (2003) for the same
vineyard. The geographic climate and zone seem to affect the
content of quercetin with respect to the same vineyard
(Gambelli and Santaroni 2004). In fact, the environmental
conditions, such as temperature, rainfall/humidity, latitude,
level above the sea, and geochemical characteristic, can affect
Table 1 Validation parameters for quercetin by HPLC-UV
Parameter Standard Matrix*
Recovery/accuracy (%) 90 ± 5 87 ± 7.0
Intra-day repeatability (RSD%) n = 10 2 8
Interlay repeatability (RSD%) n = 5 5 14
Linearity (R2) 0.9998 0.9989
LOD (mg L−1) 0.05 0.1
LOQ (mg L−1) 0.1 0.3
*BBlank^ wine, used as matrix, for the validation was a red wine free of
quercetin
 Food Anal. Methods

Table 2 Content of quercetin in some Italian red wines (mg L−1) and relative standard deviation (RSD %)

Wine/vine** Region of Italy Aging (years) Quercetin (mg L−1) Relative standard
deviation (RSD %)

R Brand 1 Chianti/Sangiovese+other Tuscany, CI 3 4.3 10.0

R Brand 2 Barbera d’Asti/Barbera +other Piemonte, NI 3 3.8 9.7
R Brand 3 Sangiovese Lazio, CI 3 0.5 5.0
R Brand 4 Sangiovese Basilicata, SI 3 0.7 6.3
R Brand 5 Sangiovese Marche, CI 3 0.3 5.8
R Brand 6 Moscato Piemonte, NI 1.5 1.1 12.0
R Brand 7 Salice Talentino/(Negroamaro+other mix) Puglia, SI 2 1.5 7.9
R Brand 8 Nero d’Avola Sicily, SI 6 0.5 10.5
R Brand 9 Nero d’Avola Sicily, SI 6 < LOQ
W Brand 10 Pinot grigio Trentino, NI 4 +
W Brand 11 Grechetto/Grechetto+other Umbria, CI 2 < LOD
W Brand 12 Verdicchio Marche, CI 3 < LOD

**If not reported differently, wine and vine denominations are identical
< LOD, below the limit of detection
All the data are the mean of nine determinations (three aliquots injected three times each)
R, red wine; W, white wine; NI, north of Italy; CI, center of Italy; SI, south of Italy
+, LOD < concentration < LOQ

the level of antioxidant compounds due to the vine maturation.
After all, the content of quercetin in brands 2 and 6, coming
from the same region (but from different grape varieties), is
quite different and brand 7 gave a quercetin content different
from that found in the same regions for different grape varie-
ties (Ragusa et al. 2017). Brand 1 was richer of quercetin than
the other brands according to Lante et al. (2003).
In addition, the possibility to determine the content of total
quercetin considering the contribution of the derivates (quer-
cetin glucosides or glucuronides) was investigated. Our intent
was to determine the sum of free and conjugated quercetin, as
free aglycone, after acidic hydrolysis, according to other au-
thors (Gambelli and Santaroni 2004; Vuorinen et al. 2000).
The results (not shown) highlighted the degradation of the
formed quercetin up to 40% in acid medium, confirming the
complexity of determining total quercetin glucosides or glu-
curonides as free aglycone. Further research are needed for
determining each single quercetin derivate in wine, in order
to better characterize its active compounds.
good agreement to those of other authors, more samples are
needed to establish if the findings of the present research de-
pend on cultivar, wine technology, climatic factors, and/or
geographical region. Moreover, stating the difficulty of deter-
mining the contribution of the conjugated quercetin, after
acidic hydrolysis, new studies by HPLC-MSMS are in prog-
ress to refine this method in order to determine the specific
amount of the single-quercetin glucosides or glucuronides and
to fix typical diagnostic ratios.
Compliance with Ethical Standards
This article does not contain any studies with human participants or an-
imals performed by any of the authors.
Conflict of Interest Francesca Buiarelli declares that she has no conflict
of interest. Flaminia Bernardini declares that she has no conflict of inter-
est. Patrizia Di Filippo declares that she has no conflict of interest.
Carmela Riccardi declares that she has no conflict of interest. Donatella
Pomata declares that she has no conflict of interest. Giulia Simonetti
declares that she has no conflict of interest. Roberta Risoluti declares that
she has no conflict of interest.

Conclusions Informed Consent Not applicable.

The present work fits in the perspective of characterizing wine

in all its Bactive^ components, in line with our previous re-
searches about this topic. In this paper, quercetin was chosen
as a marker of wine. The proposed method is simple, econom-
ic, and suitable for laboratories involved in routine analyses.
After its validation, it was applied to several commercial
Italian wines. Although chemical analysis and trend are in
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