Food Chemistry 220 (2017) 420426

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Determination of 5-hydroxymethylfurfural in honey, using headspace-

solid-phase microextraction coupled with a polyoxometalate-coated
piezoelectric quartz crystal
Marta I.S. Verssimo a,b, Jos A.F. Gamelas c, Dmitry V. Evtuguin a, M. Teresa S.R. Gomes b,
a
CICECO & Department of Chemistry, University of Aveiro, Aveiro, Portugal
b
CESAM & Department of Chemistry, University of Aveiro, Aveiro, Portugal
c
CIEPQPF & Department of Chemical Engineering, University of Coimbra, Coimbra, Portugal

a r t i c l e i n f o a b s t r a c t

Article history: High concentrations of 5-hydroxymethylfurfural (HMF) in honey provide an indication of overheating
Received 10 May 2016 under inappropriate storage conditions or aging. Conventional methods for determining HMF are
Received in revised form 5 September 2016 cumbersome and require expensive equipment or hazardous reagents. Hence the aim of this study
Accepted 29 September 2016
was to propose a new analytical tool for HMF determination in honey, using a low cost acoustic wave sen-
Available online 30 September 2016
sor. Volatile organic compounds (VOCs) of honey samples were extracted, using the solid phase microex-
traction (SPME) technique, and HMF was quantified, using a piezoelectric quartz crystal with gold
Keywords:
electrodes coated with a layer of decamolybdodivanado phosphoric acid, sensitive to HMF. The reliability
5-Hydroxymethylfurfural
Honey
of the proposed method was confirmed after comparing the results of HMF quantification with those
Acoustic wave sensor obtained by the conventional spectrophotometric White method, and no statistical differences were
SPME found (a = 0.05).
2016 Elsevier Ltd. All rights reserved.

1. Introduction cytotoxic, mutagenic, carcinogenic and genotoxic (Janzowski,

5-Hydroxymethylfurfural (HMF) is a water-soluble heterocyclic
organic compound derived from sugars, especially fructose. Very
low amounts of HMF are naturally found in fresh honey because
it is mainly formed during acid-catalysed dehydration of the
hexoses during extended food storage or as an intermediate in
the Maillard reaction during thermal treatments (Capuano &
Fogliano, 2011; Silva, Gauche, Gonzaga, & Costa, 2016). Besides
temperature, other storage conditions, such as the use of plastic
or metallic containers and moisture, contribute to HMF formation.
High HMF content in honey may also be an indication of addition
of invert syrups (Tosun, 2013). Therefore, HMF content is consid-
ered an excellent indicator of honey freshness, as well as evidence
of poor storage conditions, excessive heat-treatment or possible
adulteration with other sugars or syrups.
HMF has also been quantified in honey because it is a contam-
inant with potential harmful properties. Although it is not clear
whether human exposure to HMF presents a potential health risk
(Abraham et al., 2011; Capuano & Fogliano, 2011; Islam, Khalil,
Islam, & Gan, 2014), in vitro studies have shown that HMF is
Corresponding author.
E-mail address: mtgomes@ua.pt (M. Teresa S.R. Gomes).
Glaab, Samimi, Schlatter, & Eisenbrand, 2000; Teixid, Santos,
Puignou, & Galcetan, 2006). The Codex Alimentarius Commission
(Alinorm 01/25, 2000) established that the HMF content in honey,
after processing and/or blending, must not be higher than
80 mg kg
1. However, a European Union (Directive, 2001/110/EC)
has recommended a lower limit of 40 mg kg
1 with a few excep-
tions: 80 mg kg
1 for honey coming from regions with tropical
temperatures and 15 mg kg
1 for honey with low enzymatic levels.
The International Honey CommissionIHC (Bogdanov, 2009)
has recommended three methods for the determination of HMF:
two conventional spectrophotometric techniques (White and
Winckler methods) and liquid chromatography with UV detection
(HPLC method). Several other methods have been reported for the
analysis of HMF, employing capillary electrophoresis (Bignardi,
Cavazza, & Corradini, 2014; Biluca et al., 2014; Rizelio et al.,
2012; Silva, Schuch, Vainstein, & Jablonski, 2008), liquid
chromatography-mass spectrometry (Teixid, Moyano, Santos, &
Galceran, 2008) and gas chromatographymass spectrometry
(Gaspar & Lopes, 2009; Teixid et al., 2006; Wardencki, Chmiel,
Dymerski, Biernacka, & Plutowska, 2009). However, all these
sophisticated technologies have associated setbacks: demand the
use of expensive equipment, consume considerable amounts of
solvents or require derivatization procedures. The scientific

http://dx.doi.org/10.1016/j.foodchem.2016.09.204
0308-8146/ 2016 Elsevier Ltd. All rights reserved.
 M.I.S. Verssimo et al. / Food Chemistry 220 (2017) 420426
communitys increasing concern about the potentially toxic effects
of HMF boosts the significant efforts to establish new, rapid, low
cost and sensitive methodologies for HMF determination in
honeys.
Acoustic wave sensors, based on piezoelectric quartz crystals,
are known for their simplicity and low cost, as well as for their
reliability when carefully designed. The piezoelectric quartz crystal
vibrates with a frequency proportional to its thickness whenever
an alternate electric field is applied between the electrodes
deposited on its face. Depositing a film results in a decrease in
the frequency proportional to its mass. Sensitivity and selectivity
to an analyte are achieved by careful selection of a recognition
layer applied onto the electrode(s). Frequency of the coated crystal
becomes the sensor baseline, and the frequency decrease produced
after contact with the analyte allows sensor calibration and quan-
tification of the analyte in the sample.
Acoustic wave sensors have been successfully used by this
research group in several areas, namely sulphur and carbon diox-
ide in wine (Gomes, Rocha, Duarte, & Oliveira (1996a, 1996b),
metal analysis in food and water (Verssimo & Gomes, 2008;
Verssimo, Oliveira, & Gomes, 2007), detection of spoiled fruit
(Fernandes, Oliveira, & Gomes, 2008), halitosis evaluation
(Fernandes, Rolo, Oliveira, & Gomes, 2009), study of HPMC gelation
(Verssimo, Pais, & Gomes, 2010), and determination of pour point
of biodiesel (Verssimo & Gomes, 2011). Recently, acoustic wave
sensors have been used to detect volatile compounds from cheese,
using solid-phase microextraction (SPME) as the extraction
technique (Pais, Verssimo, Oliveira, & Gomes, 2015). The SPME
technique is quite advantageous for extracting volatiles: it elimi-
nates the use of toxic organic solvents, allows the quantification
of a large number of molecules, requires little or no preparation
of samples and, moreover, it is simple and can be easily coupled
to various analytical instruments. Several different SPME fibres
have been reported for the extraction of honey samples and, in
general, all give good results in extracting volatiles (Baroni et al.,
2006; Cuevas-Glory, Ortiz-Vzquez, Centurin-Yah, Aleac, &
Sauri-Duch, 2008; Gaspar & Lopes, 2009; Plutowska, Chmiel,
Dymerski, & Wardencki, 2011; Pontes, Marques, & Cmara,
2007), particularly the divinylbenzene/carboxen/polydimethylsi
loxane (DVB/CAR/PDMS) type. As far as we know, there has never
been a reported sensor for HMF, and therefore no reference to the
use of SPME with an acoustic wave sensor for detecting HMF in
honeys, can be found in the literature.
In this study, we propose a new method for the analysis of HMF
in honey, using an acoustic wave sensor. The SPME technique with
a DVB/CAR/PDMS fibre was applied to extract volatile compounds
from honey and an acoustic wave sensor, coated with a sensitive
layer of decamolybdodivanadophosphate (a-[PV2Mo10O40]5
),
was used for the first time to detect HMF. This Keggin-type polyox-
ometalate (POM) was previously reported as an effective redox
catalyst for volatile organic compound (VOCs) oxidation
(Gamelas, Evtyugina, Portugal, & Evtuguin, 2012), along with its
sensitivity to HMF when used as the recognition layer of the
acoustic wave sensor.
The reliability of the sensor was proved by comparing the
analytical results with those obtained by the conventional spec-
trophotometric White method (AOAC, 1990).
2. Materials and methods
2.1. Chemicals and materials
All reagents used were of analytical grade. Sodium chloride
(99.5%), used to obtain the adequate ionic strength, was supplied
by Merck (Darmstadt, Germany). Potassium hexacyanoferrate (II)
421
was from Riedel-de Han (Seelze, Germany) and zinc acetate dehy-
drate was from Panreac (Barcelona, Spain).
Analytical standard-grade 5-hydroxymethylfurfural (HMF) and
sodium bisulfite were obtained from Sigma-Aldrich (Steinheim,
Germany). Ultrapure water was produced by a Milli-Q system from
Millipore (Mississauga, Canada).
The SPME fibre used in this work was a divinylbenzene-car
boxen-polydimethylsiloxane (DVB/CAR/PDMS, 50/30 lm) type
mounted on a SPME holder for manual sampling. Both the fibre
and the holder were obtained from Supelco (Bellefonte, PA, USA).
The piezoelectric quartz crystal was coated with decamolybdo-
divanado phosphoric acid (H5[PV2Mo10O40]11H2O), Synthesized as
described previously (Gamelas et al., 2012), by spreading its etha-
nol solution (Panreac, Barcelona, Spain). Nitrogen was Alphagaz
from ArLquido. Raman spectra of gold-coated quartz crystal, with
or without POM, were recorded on a BRUKER RFS 100/S Raman
spectrometer with a YAG:Nd laser (1064 nm) under backscattering
configuration. The spectral range was 2001800 cm
1 and resolu-
tions of 4 cm
1. 2000 scans were collected to obtain a reasonable
signal-to-noise ratio. Magnesium perchlorate, used in a desiccant
column, was from Riedel-de Han (Seelze, Germany).
2.2. Sample and solution preparations
Locally produced honey (LPH) samples, collected by small local
producers in the central region of Portugal, (Leiria, Aveiro, Mira and
Viseu) after diverse periods of storage, without or after heat treat-
ment, as well as samples from commercial honeys (CH) of several
brands, which were produced in different regions but purchased
this year, 2016, and within the sell-by date, were analysed. A stan-
dard solution of 10,000 mg l
1 of 5-hydroxymethylfurfural was
prepared (weekly) by dissolving in the appropriate amount in
Milli-Q water, and stored at 4 C.
Solutions for the spectrophotometric method were prepared
according to the AOAC Official Method (AOAC, 1990): Carrez solu-
tion I was prepared by dissolving 15 g K4Fe(CN)63H2O and diluting
to 100 ml with Milli-Q water; Carrez solution II was prepared by
dissolving 30 g of Zn(CH3COO)22H2O and diluting to 100 ml with
Milli-Q water; Sodium bisulfite solution (0.20%) was prepared daily
by dissolving 0.20 g of NaHSO3 and diluting to 100 ml with Milli-Q
water.
2.3. Set-up for the acoustic wave sensor methodology
Fig. 1a shows a typical representation of a piezoelectric crystal.
In this work a 9 MHz quartz crystal, AT-cut, HC-45 with gold
electrodes was used. The crystal was coated by depositing a drop
of a solution of decamolybdodivanado phosphoric acid (POM) dis-
solved in ethanol, as shown in Fig. 1b. Solvent was then allowed to
evaporate until the frequency of oscillation of the quartz crystal
became stable. A frequency decrease of 51 kHz was obtained. The
limit of detection and sensitivity of the sensor depend on the coat-
ing amount, which must not exceed the limit at which vibration is
impaired, but must be high enough to assure the sensitivity neces-
sary for quantitation.
The cell housing the piezoelectric crystal has already been
described (Fernandes et al., 2009), and the setup is similar to the
one reported by Gaspar and Gomes (2012), except for a desiccant
column that was included prior to the sensor cell. Briefly the
coated piezoelectric quartz crystal was housed inside a PVC cell
with two inlets and an exhausting outlet. A small oven was used
for desorbing the compounds from the SPME fibre. A nitrogen flow,
controlled by a flowmeter (Cole Parmer, Illinois, USA), carried the
desorbed volatile compounds from the fibre and directed them to
the centre of both faces of the sensor, after passing through a
desiccant column to remove water. The piezoelectric sensor was
422 M.I.S. Verssimo et al. / Food Chemistry 220 (2017) 420426
Fig. 1. (a) Piezoelectric quartz crystal with gold electrodes and (b) coating process by drop deposition.
connected to a laboratory-made oscillator, which in turn was con-
nected to a frequency counter (Leader LF-827). Frequency data
were monitored and stored in a PC connected through a GPIB to
the frequency meter.
2.4. Procedure for HMF determination, using the acoustic wave sensor
2.4.1. Sample preparation and extraction of volatile HMF from honey,
using headspace-solid-phase microextraction (HS-SPME)
Before the first analysis of the day, the fibre was cleaned for
15 min at 260 C. After this initial cleaning, and between analyses,
the fibre was always kept in an empty vial filled with nitrogen in
order to protect it from VOCs from the environment.
HMF determination in honey samples was performed by the
standard additions method, intended to eliminate matrix effects.
Equal weights of honey were spiked with known but different
amounts of HMF, as below. One gramme of a honey sample was
weighed into each one of the six 4 ml vials, and diluted (1:1) with
Milli-Q water, in order to decrease the density of the sample in an
attempt to allow stirring of the sample with a magnetic bar, facil-
itating the escape of volatiles to headspace (Cuevas-Glory et al.,
2008). Extraction efficiency was also improved by increasing ionic
strength, by adding sodium chloride (30% w/v), which decreases
the solubility of hydrophilic compounds in the aqueous phase
(Pontes et al., 2007). A known amount (between 0 and 150 ll) of
the 10,000 mg l
1 HMF standard solution was added to each vial,
along with the magnetic stirrer, after which the vial was sealed
with a screw cap equipped with a silicone septum. The vial which
is about to be analysed was then kept in a water bath at 60 C for
temperature equilibration (15 min).
Fig. 2 presents, step-by-step, the analysis process. The SPME
fibre coated with DVB/CAR/PDMS was introduced into the head-
space through the septum, and exposed for exactly 45 min, while
stirring velocity was controlled. After the extraction step, the fibre
was removed from the vial and inserted into the injection port of
the homemade oven set at 260 C for thermal desorption.
2.4.2. Desorption and analysis of HMF, using an acoustic wave sensor
The analytical system was conceived for a flow injection
methodology. There was an injection port on the top of a small
oven whose outlet led to the sensor cell. Nitrogen flowed through
the system at a flow rate adjusted to 54 ml min
1. Daily, the desic-
cant column (with magnesium perchlorate) that is intended to dry
the gases entering the crystal cell, was renewed. The acoustic wave
sensor was left to stabilize, and baseline frequency was attained,
while the SPME fibre was extracting the volatile compounds from
the honey sample headspace. Afterwards, the SPME fibre was intro-
duced, through the injection port, inside the oven at 260 C, and the
desorbed volatiles were carried by the nitrogen flow towards both
faces of the sensor. The frequency of the piezoelectric crystal began
to decrease, due to the interaction of the desorbed compounds with
the POM coating, after which it began to increase again, as HMF was
being desorbed, and swept away by the constant nitrogen flow. The
minimum frequency value was recorded and the difference to the
baseline frequency computed. The system was ready for a new injec-
tion after the frequency reached the baseline value, evidence that
complete recovery of the sensor was attained.
2.5. Procedure for HMF determination, using the spectrophotometric
conventional method (White method)
The White method (AOAC, 1990) involves measuring the absor-
bance of a clarified aqueous honey solution against a reference
solution of the same honey in which the 284 nm chromophore of
HMF was destroyed by bisulfite salt.
Accurately weighed amounts of five grammes of honey were
dissolved in 25 ml of Milli-Q water and transferred quantitatively
into a 50 ml volumetric flask. 0.5 ml of Carrez solution I was added
to the sample and mixed well. 0.5 ml of Carrez solution II was then
added and again mixed well. The sample was brought to a final vol-
ume of 50.0 ml with Milli-Q water. The solution was filtered and
the first 10 ml of filtrate were discarded. Aliquots of 5.00 ml were
transferred into two test tubes: to one tube, 5.00 ml of distilled
water were added (sample solution); to the second tube, 5.00 ml
of 0.2% sodium bisulfite solution were added (reference solution).
Both sample tubes were well mixed in a vortex mixer (VWR,
Radnor, PA, USA).
The absorbance of the solution was measured against the refer-
ence at 284 nm and 336 nm, using a double beam Hitachi U-2000
UVvis Spectrophotometer. HMF content in honey was then
calculated, using the following equation (AOAC, 1990):
HMFmg=100 g of honey A284
A336  Factor=W
where:
W = weight of sample in grammes.
Factor = (126  100  1000  100)/(16,830  1000) = 74.87
126 = molecular weight of HMF
16,830 = molar absorptivity of HMF at 284 nm
 M.I.S. Verssimo et al. / Food Chemistry 220 (2017) 420426 423

Fig. 2. The new methodology step-by-step: equilibration, extraction, desorbing, detection and frequency data storage.

3. Results and discussion
3.1. Acoustic wave sensor assembly
Acoustic wave sensor assembly was done by depositing the syn-
thesized POM from its ethanol solution on the surface of the gold-
coated quartz crystal. The presence of POM on the gold electrode,
after ethanol evaporation, was confirmed while comparing the
Raman spectra of electrode before and after the POM deposition
(Fig. S1 in Supplementary Data). Thus Raman spectra clearly
showed the characteristic signals at 1054 and 993 cm
1 belonging
to stretching in the V@O and Mo@O of VO4 and MoO4 structural
groups of POM, respectively. Additionally, the signals detected at
835/768 and 247 cm
1 were assigned to stretching and bending
modes, respectively, in MoOMo bridges, and are typical of this
Keggin-type POM (Molinari, Nakka, Kim, & Wachs, 2011).
In the analytical method using the acoustic wave sensor (Fig. 2),
volatiles sorbed onto the SPME fibre during the extraction step
were injected inside the oven at 260 C and the desorbed com-
pounds were carried by the nitrogen flow towards both faces of
the sensor. The frequency of the piezoelectric crystal decreased,
due to the interaction of the desorbed HMF with the POM coating
layer, after which it began to increase again until it reached the
baseline value. The plausible triggering mechanisms may include
the redox interaction of HMF with POM, thus forming an interme-
diate adduct via the electron transfer complex. Such interactions of
aldehydes with this kind of POM are rather common and were
widely reported for both homogeneous and heterogeneous cat-
alytic reactions (Khenkin, Weiner, Wang, & Neumann, 2001;
Kozhevnikov, 1998). The confirmation of eventual HMF oxidation
by POM was evidenced by the changing of the POM colour from
orange (characteristic for V(V)) to blue-green (characteristic for V
(IV)) while injecting HMF into the nitrogen stream (Fig. 2). The oxi-
dation of V(IV) back to V(V) in POM, after further elution with
nitrogen, was also confirmed by UVvis analysis (results not
shown), thus demonstrating the reversibility of POM redox process
under the analysis conditions. However, the mechanisms involved
in interaction between HMF and POM need further studies.
(equilibration and extraction). The effect of adding sodium chloride
(30%) to the sample vials was also studied, and an increase in
extraction was confirmed. Fig. 3 shows the response obtained by
the acoustic wave sensor for a honey sample spiked with 40 lg
of HMF/g of honey, which corresponds to the limit allowed by leg-
islation (Directive, 2001/110/EC). It can be seen that frequency
starts decreasing as soon as HMF reaches the crystal and interac-
tion with the crystal coating begins. This interaction is reversible,
and the frequency of oscillation increases as the nitrogen stream
carries the compound away, until baseline frequency is restored.
Complete recovery of the sensor was accomplished within a few
minutes.
A honey sample freshly harvested obtained from a trustable
source, and therefore presumed to be as HMF-free as possible,
and to which the sensor did not respond significantly, was used
to calculate the limit of detection (LOD) and limit of quantification
(LOQ) for the SPME/acoustic sensor methodology. LOD and LOQ
were calculated from the intercept (x0) of the standard addition
calibration line, and syx that measures the variability, or scatter
of the observed values around the regression line. The LOD (con-
centration corresponding to a signal = x0 + 3syx) was 3.4 lg g
1,
while the LOQ (concentration corresponding to a signal =
x0 + 10syx) was 11.4 lg g
1, which is below the EU guideline limit
for HMF in honey.

3.2. Assessment of the new acoustic wave methodology for HMF

determination in honey

Initial studies showed that SPME extraction was more efficient

if magnetic stirring was maintained during all of the process Fig. 3. Frequency shift (Hz) caused by a spiked (40 lg of HMF) honey sample.
424 M.I.S. Verssimo et al. / Food Chemistry 220 (2017) 420426

calibration lines prepared with a delay of one week, from samples

with HMF contents below LOD, and corresponding to additions of
the same quantities, were compared by the paired t-test, and no
significant differences were found (a = 0.05).

3.3. HMF content, its link to consumers routines, and the importance
of conclusion dissemination

From Table 1 it is possible to observe that HMF values for LPH1

Fig. 4. Frequency decreases (Hz) vs. HMF content (lg g
1) for LPH2.
The LOD and LOQ for the spectrophotometric method were
calculated from the mean and the standard deviation of blanks.
LOD (concentration corresponding to a signal = xblank + 3sblank)
was 3.3 lg g
1, while the LOQ (concentration corresponding to a
signal = xblank + 10sblank) was 7.3 lg g
1. Magnitudes of both LOD
and LOQ were of the same order as those in the new proposed
methodology.
Fig. 4 shows the calibration curve obtained during the analysis
of a sample (LPH2), by the new sensor and using the standard addi-
tion method. The HMF concentration in honey could be obtained
by extrapolation of the regression line. Locally produced honey
(LPH) and commercial honey (HC) were analysed, both by the
new methodology, using the SPME technique with the acoustic
wave sensor and by the White method (spectrophotometric).
Table 1 shows the results obtained for both methodologies. For
the acoustic wave method, HMF concentration was obtained using
the standard addition method with a calibration curve of six
points, while, for the spectrophotometric method, the results were
the average of five replicates.
The values in Table 1, obtained with the two methodologies,
were statistically compared, using a paired t-test, and it was found
that the difference in the mean values between the results of the
two methods was not great enough to exclude the possibility that
the difference was due to random variability. An F-test was run for
the comparison of standard deviations and, for all samples except
one, the proposed method was more precise than was the standard
method (a = 0.05).
Due to the nature of the samples, with very different matrices
(viscosity, texture and granulometry), and much difference from
external standards prepared in distilled water, the proposed
methodology is based on standard additions, and therefore, a fresh
calibration was prepared for each sample. However, y values of
and LPH2 stand out. Honey samples for LPH1 and LPH2 were taken
from the same jar, but from different locations. Having been stored
for more than 15 years at temperatures between 10 and 15 C, this
honey jar showed two distinct layers: the bottom layer appeared to
be more crystalized and opaque (LPH2), while the upper layer was
more liquid and dark (LPH1). The HMF content found for both lay-
ers was higher than 40 lg g
1, the allowed limit. The upper layer
(LPH1) showed the higher result, 78.3 0.1 lg g
1, while the bot-
tom layer (LPH2) showed an HMF content of 71.5 0.1 lg g
1.
The appearance of the two layers for this unprocessed honey can
be explained by the spontaneous crystallization that occurs in hon-
eys with long storage periods. The difference in HMF content of the
two phases (the liquid phase on top and a more solid phase at the
bottom) is expected and can be explained by water activity. In the
liquid phase, water activity becomes higher than that in the
original honey. This is accompanied by a decrease in the solution
concentration due to the release of water from the solid phase as
a consequence of crystal formation. This makes the substrate con-
ditions suitable for the development of the microbial flora and,
within time, produces subsequent organoleptic modifications and
quality damages, including increasing HMF content (Tosi, Lucero,
& Bulacio, 2004).
Thermal treatment of honey is sometimes necessary to sterilize
it, or to obtain a more appealing organoleptic product by reducing
its viscosity in order to facilitate the dosage and jar filling, to avoid
post-bottling crystallization or delay its appearance (Cozmuta,
Cozmuta, Varga, Marian, & Peter, 2011). In Table 1, honey samples
CH2, CH4 and CH5 correspond to the same honey, subjected to
different thermal treatments. CH2, which is the original honey
without thermal treatment, presented an HMF content below the
detection limit of both methodologies, while CH4, the same honey
after being at 40 C for 64 h, showed an HMF content of
14.6 0.3 lg g
1. CH5, the same honey after being heated to
80 C for 10 min, showed a higher content of HMF,
18.8 0.1 lg g
1. These results confirm as expected, that HMF for-
mation is influenced by temperature and heating time. Besides, it
can be concluded that even mild conditions, e.g. 40 C, do increase
HMF content.
LPH4 and LPH5 were obtained from a family which had received
them as gifts from producing friends. While LPH4, which is an
older sample not consumed due to its taste (heather) that did

Table 1
HMF content (lg g
1) in locally produced (LPH) and commercial (CH) honeys.

Age, vegetal origin, place, treatment Acoustic sensor [HMF] lg g
1 White method [HMF] lg g
1
Locally produced honey
LPH1 >15 years, flowers and fruits, Leiria, none 78.3 0.1 77.9 1.0
LPH2 >15 years, flowers and fruits, Leiria, none 71.5 0.1 71.2 0.7
LPH3 <1 year, flowers, Anadia, none <LOD <LOD
LPH4 >5 years, dark, heather, Viseu, none 18.5 0.4 18.2 0.9
LPH5 <1 year, flowers, Mira, heated 15 min/water bath 41.8 0.2 41.0 1.0
Commercial honey
CH1 Within sell-by date, rosemary, mountainous interior region, none 25.7 0.4 25.5 0.8
CH2 Within sell-by date, orange blossom, Penamacor none <LOD <LOD
CH3 Within sell-by date, heather, Serra da Malcata, none 8.4 0.8 8.9 0.7
CH4 Within sell-by date, orange flower, Penamacor, 64 h/40 C 14.6 0.3 14.4 0.8
CH5 Within sell-by date, orange flower, Penamacor, 10 min/80 C 18.8 0.1 18.6 0.8
 M.I.S. Verssimo et al. / Food Chemistry 220 (2017) 420426
not please the children, showed a result of 18.5 0.4 lg g
1, LPH5,
although being more recent (2015), showed a much higher HMF
content of 41.8 0.2 lg g
1. Besides being from a different origin,
it is not foreseeable that this is the reason for such a high content,
as other honey, from not a very distant region and of the same age
(LPH3), did show a value below the detection limit. The explana-
tion must be the heating for 15 min in a boiling water bath. This
procedure was normal for this family, in order to obtain a more
appealing product. However, its HMF value, which is above the
regulated limit, signifies the need to spread information about
the risks of HMF and how it is formed.
4. Conclusion
A new methodology for HMF determination in honeys is pre-
sented and compared with the conventional spectrophotometric
method. This method combines the great effectiveness of SPME
fibres for extracting volatiles from honey with the high sensitivity
of the low-cost piezoelectric quartz crystals coated with
decamolybdodivanadophosphate. The use of the standard addi-
tions method to eliminate the matrix effect of honey, and the use
of the decamolybdodivanado phosphoric acid as a sensitive layer
to HMF, allowed quantifying HMF in honey samples. Sensor life
was at least 6 weeks, without sensitivity loss, and quantification
limit was well below the legislation threshold. Comparing the
results obtained after analysing honey samples by the sensor and
the conventional spectrophotometric methodology, showed no
statistically significant differences (a = 0.05) in HMF content,
although precision was 9 times out of ten better with the sensor.
Conflict of interest
The authors declare no competing financial interest.
Acknowledgements
This work was developed within the scope of the project
CICECO, POCI-01-0145-FEDER-007679 (FCT Ref. UID/CTM
/50011/2013), financed by national funds through the FCT/MEC
and when appropriate co-financed by FEDER under the PT2020
Partnership Agreement. Thanks are also due, for financial support,
to CESAM (UID/AMB/50017), to FCT/MEC through national funds,
and the co-funding by the FEDER, within the PT2020 Partnership
Agreement and Compete 2020. M. Verssimo would like to
acknowledge the Portuguese Foundation for Science and Technol-
ogy (FCT), for the post-doctoral grant BPD/30134/2006.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2016.
09.204.
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Marta I.S. Verssimo has a post-doc position at the University of Aveiro. She
received a BS degree in Chemistry in 1998 from the University of Aveiro and a PhD
in analytical chemistry in 2003 from the same University. Her current research
interests are in chemical sensors and fibre optic sensors.
Jos A.F. Gamelas is member of the Chemical Process Engineering and Forest
Products Research Centre. He received his BS degree in analytical chemistry from
the University of Aveiro in 1992 and a PhD in chemistry from the same University in
2001. His current research interests are related to polyoxometalates applications,
cellulose modification, and inverse gas chromatography applied to the materials
surface chemical characterization.
Dmitry V. Evtuguin graduated in Chemical Engineering and Technology in 1985 by
the Leningrad Forest Technical Academy (LFTA, USSR), and got his PhD in Chemistry
by the same institution in 1988. Associate Professor at the Department of Chemistry
of the University of Aveiro since 1995. Author/co-author of more than 200 articles
and book chapters, 260 conference communications and of six patents in the areas
of lignocellulosics chemistry and technology and redox catalysis, including catalysis
by polyoxometalates.
M. Teresa S.R. Gomes is an associate professor at the University of Aveiro. She
received her BS degree in chemical engineering from the University of Coimbra in
1983 and a PhD in analytical chemistry from the University of Aveiro in 1997. Her
current interests are chemical sensors and analytical chemistry.