ORIGINAL PAPER
Analysis of flavonoids in honey by HPLC coupled
with coulometric electrode array detection and electrospray
ionization mass spectrometry
Karoline Petrus & Heidi Schwartz & Gerhard Sontag
Received: 4 November 2010 /Revised: 12 December 2010 /Accepted: 15 December 2010 /Published online: 13 January 2011
# Springer-Verlag 2011
Abstract The analysis of flavonoids in unifloral honeys by
high-performance liquid chromatography (HPLC) coupled
with coulometric electrode array detection (CEAD) is
described. The compounds were extracted by a nonionic
polymeric resin (Amberlite XAD-2) and then separated on
a reversed phase column using gradient elution. Quercetin,
naringenin, hesperetin, luteolin, kaempferol, isorhamnetin,
and galangin were detected in a coulometric electrode array
detection system between +300 and +800 mV against
palladium reference electrodes, and their presence was
additionally confirmed by HPLC coupled with electrospray
ionization mass spectrometry. The method was applied to
analysis of 19 honeys of different varieties and origin. The
limits of detection and quantitation ranged between 1.6 and
8.3 μg/kg and 3.9 and 27.4 μg/kg, respectively. The
recoveries were above 96% in fluid and above 89% in
creamy honeys. Some of these honeys (melon, pumpkin,
cherry blossom, dandelion, maple, and pine tree honey)
were investigated for their flavonoid content and profile for
the first time. Differences between honeys were observed
both in flavonoid concentrations and in the flavonoid
profiles. The flavonoid concentrations ranged from 0.015
to 3.4 mg/kg honey. Galangin, kaempferol, quercetin,
isorhamnetin, and luteolin were detected in all investigated
honeys, whereas hesperetin occurred only in lemon and
orange honeys and naringenin in lemon, orange, rhododen-
dron, rosemary, and cherry blossom honeys.
Keywords Honeys . Flavonoid profiles . HPLC-CEAD .
HPLC-ESI-MS
Introduction
A wide range of minor constituents like phenolic acids,
flavonoids, certain enzymes, carotenoid-like substances,
vitamins, organic acids, and Maillard products are present in
honey. The composition is rather variable and primarily
depends on the floral source. Among other parameters,
flavonoid as well as flavonoid glucoside profiles or the
presence of a single flavonoid can contribute to identify
unifloral honeys. A great number of such honeys are on the
market and have to be checked regarding their quality,
authenticity, and labeling in order to prevent consumer
deception. Therefore, there is a necessity for analytical
methods which enable reliable evaluation of unifloral honeys.
So far, different methods have been developed for
determination of flavonoids in honey. Due to the complex
matrix of honey and the low concentration of these
compounds, numerous authors have proposed a sample
cleanup including a preconcentration step. The cleanup
depends on the question whether flavonoid aglycones or
glycosides should be analyzed. In the first case, the honey
sample is dissolved in acidified water. Then, the flavonoid
aglycones can be extracted with ethylacetate [1, 2], by solid
phase extraction on reversed phase materials [3–5] or on
nonpolar resins with [6–13] or without [14–16] an
Published in the special issue Analytical Sciences in Austria with
Guest Editors G. Allmaier, W. Buchberger, and K. Francesconi.
K. Petrus : H. Schwartz : G. Sontag (*)
Institute for Analytical Chemistry, University of Vienna,
Währinger Strasse 38,
1090 Vienna, Austria
e-mail: gerhard.sontag@univie.ac.at
H. Schwartz
Center for Analytical Chemistry,
Department of Agrobiotechnology (IFA Tulln),
University of Natural Resources and Life Sciences,
Gregor-Mendel-Straße 33,
1180 Vienna, Austria
Anal Bioanal Chem (2011) 400:2555–2563
DOI 10.1007/s00216-010-4614-7

additional extraction step with ethyl ether or 2-butanol [17]
or further purification on a Sephadex column [18]. With the
nonionic resins, Amberlite XAD-2 and XAD-4 sugars,
acids, pigments, and disturbing compounds can be more
effectively eliminated than on reversed-phase cartridges.
Additionally, the aglycones, being less polar than glyco-
sides, are effectively bound on the resins. Only in some
papers no extraction step was applied, and the flavonoids
were determined by analyzing the compounds after disso-
lution of the honey in pure or acidified water [19, 20]. As a
consequence of the high concentration of polar and ionic
compounds, contamination of the analytical column has to
be expected if numerous honey samples are analyzed. For
analysis of flavonoid glycosides, the sample was dissolved
in pure water, and the glycosides were pre-concentrated on
a C18-SPE-cartridge and eluted with methanol [21, 22]. For
determination of glycosides, the extraction on reversed
phase material is more appropriate than the extraction on
nonpolar resins.
Due to the polarity of the compounds, it is not surprising
that GC with flame ionization [17] or mass spectrometric
detection [23, 24] was applied only occasionally. Capillary
electrophoresis with UV [25–27] or mass spectrometric
detection [28] has the advantage of high separation
efficiency and shorter analysis time in comparison to
high-performance liquid chromatography (HPLC). Never-
theless, it was difficult to separate all flavonoids in one run,
and it was necessary to wash the capillary after each
analysis in order to attain a better reproducibility [25]. Still,
these methods are well suited for qualitative analysis of
flavonoids in honey and propolis [25, 26, 28, 29].
The method of choice for qualitative and quantitative
analysis of flavonoids is HPLC coupled with UV [1, 3, 6, 7,
9–18, 30–38], electrochemical [20], or mass spectrometric
detectors [2, 4, 5, 19, 21, 22, 39–41]. Isocratic elution of
phenolic compounds in honey extracts by HPLC/UV
suffered from co-elution of compounds, and as a result of
the lack of selectivity, complex chromatograms were
obtained. Therefore, gradient elution was proposed. Never-
theless, UV detection is not sensitive enough to determine
trace amounts of some flavonoids. Using ultra-performance
liquid chromatography coupled with time of flight mass
spectrometry (UPLC-Q/TOF-MS), more phenolic com-
pounds could be identified compared to diode array
detection [2]. HPLC-MSn on an ion trap mass spectrometer
is a promising technique in the determination of the floral
origin of unifloral honeys, especially when the flavonoid
glycoside profiles should be investigated [21]. HPLC-
electrospray ionization (ESI/MS) has proved as a valuable
method for qualitative and quantitative assay of some
flavonoids in propolis [42].
Liang et al. have shown [20] that electrochemical
detection of phenolic compounds in citrus honey is six to
14 times more sensitive than diode array detection. Inoue et
al. [43] investigated the radical scavenging activity caused
by phenolic compounds with HPLC coupled with coulo-
metric electrode array detection (CEAD) without using the
high selectivity of this method for determination of single
phenolic compounds. Already in 1993, Jörg et al. [44] have
demonstrated that HPLC/CEAD is—due to its selectivity
and sensitivity—well suited for determination of phenolic
acid esters in different honeys with detection limits between
0.1 and 1 μg/kg.
The objective of the present study was to compare SPE
extraction of flavonoids in honey with accelerated solvent
extraction (ASE) and to develop a method based on HPLC/
CEAD for qualitative and quantitative determination of
flavonoids in different honeys. Additionally, to verify the
presence of the flavonoids, these compounds should be
identified by HPLC/ESI-MS. Some not so well known
unifloral honeys like those of melon, maple, pumpkin,
cherry blossom, dandelion, and one nonfloral honey (pine
tree) should be investigated with this method.
Materials and methods
Collection of honey samples
Honey samples (Table 1) of various floral sources were
collected in different countries from 2006 to 2008.
Chemicals and solutions
Galangin, hesperetin (purity≥95%), isorhamnetin (purity≥95%),
kaempferol (purity ≥96%), luteolin (purity ≥99%), myricetin
(purity ≥96%), and quercetin dihydrate (purity ≥99%)
were purchased from Sigma-Aldrich (Buchs, Switzerland).
Methanol, HiPerSolvChromanorm gradient grade, was from
VWR (Vienna, Austria); ethyl acetate (Rotisolve® HPLC
grade) and acetic acid (Rotipuran 100% p.a.) were from Carl
Roth (Karlsruhe, Germany). Ethanol (p.a.) was purchased
from AustrAlco (Öster. HandelsgesmbH, Spillern, Austria);
hydrochloric acid (37% p.a.), sodium hydroxide (p.a.),
potassium dihydrogenphosphate (p.a.), and ortho-phosphoric
acid (85% p.a.) were from Merck (Darmstadt, Germany); and
Amberlite XAD-2 was obtained from Supelco (Vienna,
Austria). Bi-distilled water was used in all experiments.
Stock solutions Four milligrams of each flavonoid was
dissolved in 10 ml of methanol by sonication. The solutions
were stored in dark flasks at 4 °C.
Standard mixture Fifty microliters of each stock solution
were pipetted into a 20-ml flask and filled up with
methanol, yielding 1 mg/L of each compound. This
2556 K. Petrus et al.