Methods for the determination of HMF in honey: a comparison

M. Zappal
a a, B. Fallico a,*
, E. Arena a, A. Verzera b

a
Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari (DOFATA), Facolt a di Agraria, Universit
a di Catania, Via S. Sofia, 98,
Catania 95128, Italy
b
a di Messina, Papardo, Messina 98168, Italy
Dipartimento di Chimica Organica e Biologica, Universit
Received 18 May 2003; received in revised form 3 March 2004; accepted 5 March 2004

Abstract
HMF (5-hydroxymethylfurfuraldehyde) is essential to evaluate the conformity of honey to the current legislation. Elevated
concentrations of HMF in honey provide an indication of overheating, storage in poor conditions or age of the honey. Both the
Codex Alimentarius Commission (Alinorm 01/25, 2000) and the European Union (Directive 110/2001) established that its con-
centration in honey usually should not exceed 80 or 40 mg/kg, respectively. The International Honey Commission recommends three
methods for the determination of HMF: two spectrophotometric methods, determination after White and after Winkler and a
HPLC method. These methods were recently tested by the International Honey Commission (1999). Aim of this research was to
compare HMF values in unifloral honeys measured by the three methods. From our data, HPLC and White methods usually give
similar values, except for eucalyptus honey; Winkler method gave for all honeys higher values than other two methods.

2004 Elsevier Ltd. All rights reserved.

Keywords: HMF (5-hydroxymethylfurfuraldehyde); Unifloral honey; Analytical methods

1. Introduction pean Union (EU Directive 110/2001) fixed a HMF limit

HMF (5-hydroxymethylfurfuraldehyde) measure-
ment is used to evaluate the quality of honey; generally
not present in fresh honey, its content increases during
conditioning and storage. Honey processing, requires
heating both to reduce viscosity, and to prevent crys-
tallisation or fermentation (Singh, Singh, Bawa, & Sek-
hon, 1988) in air ventilated chambers, at 45–50 C for 4/7
days or by immersion of honey drums in hot water.
Heating of unifloral honey leads to different HMF levels
in honey (Fallico, Zappala, Arena, & Verzera, 2004).
HMF is formed during acid-catalysed dehydration of
hexoses (Belitz & Grosch, 1999) and, it is connected to
the chemical properties of honey, like pH, total acidity,
mineral content (Anam & Dart, 1995; Bath & Singh,
1999; Hase, Suzuki, Odate, & Suzuki, 1973; Singh &
Bath, 1997, 1998).
Codex Alimentarius (Alinorm 01/25 2000) established
that the HMF content of honey after processing and/or
blending must not be higher than 80 mg/kg. The Euro-
*
Corresponding author. Tel.: +39-957580214; fax: +39-957141960.
E-mail address: bfallico@unict.it (B. Fallico).
0956-7135/$ - see front matter
2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2004.03.006
in honey of 40 mg/kg with the following exceptions: 80
mg/kg for honey coming from Countries or Regions
with tropical temperatures, 15 mg/kg for honey with low
enzymatic level (8-3 Schade Units).
The International Honey Commission (IHC, Stefan
Bogdanov, 1999, pp. 1–54) recommends three methods
for the determination of HMF. These methods include
two spectrophotometric methods widely used in routine
analysis, determination after White (1979) and after
Winkler (1955), as well as the HPLC. The method de-
scribed by White involves measurement of UV absor-
bance of clarified aqueous honey solutions with and
without bisulphite while that of Winkler involves mea-
surement of the UV absorbance of honey solutions with
added barbituric acid and p-toluidine (IHC, Stefan
Bogdanov, 1999, pp. 1–54).
The HPLC methods is according to Jeuring and
Kuppers (1980): honey is simply dissolved in water and,
after a filtration, HMF is determined on a reverse phase
HPLC column by isocratic elution with water and
methanol as mobile phase. HPLC separates HMF from
other components and thus avoid interference in the
determination. These interferences could be due to
various aldehydes present in the honeys according to
274 a et al. / Food Control 16 (2005) 273–277
M. Zappal

their floral origin or to products that appear during
conditioning or storage (Wootton & Ryall, 1985).
However, the use of different analytical methods for
HMF determination and the use of inaccurate or inad-
equate procedures are actually a problem. The methods
were recently tested by the International Honey Com-
mission (IHC, Stefan Bogdanov, 1999, pp. 1–54), the
methods yielded comparable values in collaborative
studies on three honey samples having different HMF
content, to cover the main range of determination. Small
differences between the methods resulted only at very
low levels, of no interest for assessing honey quality.
Aim of this research was to compare the HMF level in
unifloral honeys measured by the three methods.
2. Material and methods
2.1. Samples
and the corresponding moisture content (%) was cal-
culated according to AOAC (1980);
• electrical conductivity was measured at 20 C in a
20% (w/v) solution (dry matter basis) in deionised
water (Loveaux, Pourtallier, & Vorwohl, 1973) by a
Delta Ohm HD 8706 conductivity meter;
• ash was indirectly determined using the measured
electrical conductivity and applying the following
equation: X1 ¼ ðX2
0:143Þ=1:743 were: X1 ¼ ash
value; X2 ¼ electrical conductivity in lS/cm at 20 C
(Piazza, Accorti, & Persano Oddo, 1991);
• free acids, lactones, total acidity and pH were mea-
sured using a Mettler Toledo MP 220 pH meter
according to Official Method (Repubblica Italiana:
GU. no. 282, 12/10/1984);
• diastase determinations were conducted by an enzy-
matic-spectrophotometric method, using a kit Phade-
bas Amylase Test (Pharmacy & Upjohn Diagnostic
AB).

Fourteen different honey samples, belonging to the 2.3. HMF determination

following floral origin: acacia, citrus, eucalyptus, chest-
nut and wildflower as declared in the label of the pro-
ducer, were purchased in some local shopping centres.
The unifloral honey samples were collected as follow:
two different samples of acacia, eucalyptus and chestnut
honey and four different samples of citrus and wild-
flower honey, respectively (Table 1). Each honey sample
was purchased in duplicate in pot of 500 g.
2.2. Chemical analyses
The following chemical determinations were carried
out on honey samples:
• moisture was determined measuring the refractive
indices at 20 C by a Carl Zeiss 16531 refractometer
2.3.1. HPLC method
Five grams of honey samples were diluted up to 50 ml
with distilled water, filtered on 0.45 lm filter and
immediately injected in a HPLC (Varian 9012Q)
equipped with a Diode Array Detector (Varian, Star
330). The HPLC column was a Merck Lichrospher, RP-
18, 5 lm, 125 · 4 mm, fitted with a guard cartridge
packed with the same stationary phase (Merck, Milan).
The HPLC conditions were the following: isocratic
mobile phase, 90% water at 1% of acetic acid and 10%
methanol; flow rate, 0.7 ml/min; injection volume, 20 ll.
All the solvents were HPLC grade (Merck, Milan). The
wavelength range was 220–660 nm and the chromato-
grams were monitored at 285 nm. HMF was identified
by splitting the peak in honey with a standard HMF

Table 1
Characterisation of the different honey samples analysed
Samples Moisture (%) Ash (g%) Electrical pH Diastase Free acids Lactones Total acidity
conductivity activity (meq/kg) (meq/kg) (meq/kg)
(ms/cm) (Schade)
Acacia 1 17.4 ± 0.01 0.003 ± 0.001 0.15 ± 0.001 3.38 ± 0.02 7.7 ± 0.42 19.9 ± 0.48 4.2 ± 0.38 24.1 ± 0.51
Acacia 2 17.0 ± 0.01 n.d. 0.13 ± 0.002 3.55 ± 0.03 14.8 ± 0.38 13.3 ± 0.29 4.3 ± 0.82 17.6 ± 0.93
Citrus 1 16.6 ± 0.01 0.046 ± 0.002 0.22 ± 0.003 3.46 ± 0.01 7.8 ± 0.02 26.3 ± 0.35 4.2 ± 0.46 30.4 ± 0.11
Citrus 2 17.2 ± 0.01 0.120 ± 0.001 0.35 ± 0.001 3.46 ± 0.01 7.9 ± 0.30 29.8 ± 0.35 3.9 ± 0.92 33.6 ± 1.27
Citrus 3 19.1 ± 0.14 0.047 ± 0.001 0.23 ± 0.002 3.49 ± 0.03 10.0 ± 0.69 26.0 ± 0.01 5.0 ± 0.32 31.0 ± 0.91
Citrus 4 19.5 ± 0.01 0.050 ± 0.004 0.23 ± 0.008 3.43 ± 0.01 12.0 ± 0.93 27.0 ± 0.01 4.7 ± 0.82 31.7 ± 0.82
Eucalyptus 1 15.5 ± 0.17 0.209 ± 0.002 0.51 ± 0.004 3.68 ± 0.01 18.2 ± 0.65 29.3 ± 0.50 4.8 ± 0.84 34.1 ± 1.08
Eucalyptus 2 16.5 ± 0.35 0.235 ± 0.001 0.55 ± 0.002 3.66 ± 0.01 27.9 ± 1.18 34.1 ± 0.25 4.5 ± 0.53 38.6 ± 0.36
Chestnut 1 18.0 ± 0.12 0.688 ± 0.002 1.34 ± 0.004 4.98 ± 0.02 15.7 ± 0.63 17.3 ± 0.29 6.5 ± 0.53 23.7 ± 0.60
Chestnut 2 17.9 ± 0.12 0.929 ± 0.006 1.59 ± 0.002 5.84 ± 0.04 22.2 ± 0.94 11.4 ± 0.25 5.2 ± 0.53 16.5 ± 0.36
Wildflower 1 17.1 ± 0.10 0.129 ± 0.001 0.37 ± 0.001 3.92 ± 0.08 20.0 ± 0.31 24.1 ± 0.85 5.5 ± 0.84 29.6 ± 0.94
Wildflower 2 15.8 ± 0.01 0.061 ± 0.01 0.25 ± 0.001 3.70 ± 0.03 18.6 ± 0.29 22.9 ± 0.25 6.0 ± 2.08 28.8 ± 2.16
Wildflower 3 16.9 ± 0.06 0.550 ± 0.004 1.10 ± 0.008 4.38 ± 0.03 18.9 ± 0.49 32.5 ± 0.41 6.1 ± 0.84 38.6 ± 1.04
Wildflower 4 18.0 ± 0.10 0.178 ± 0.002 0.45 ± 0.004 3.76 ± 0.04 13.7 ± 0.49 38.3 ± 0.29 5.2 ± 0.53 43.4 ± 0.76
 a et al. / Food Control 16 (2005) 273–277
M. Zappal
(Sigma-Aldrich, Milan), and by comparison the spec-
trum of HMF standard with that of honey samples. The
amount of HMF was determined using an external
calibration curve, measuring the signal at k ¼ 285 nm.
Five grams of honey resulted the optimal weight
using the Ingamells e Switzer equation (Mannino, 2001).
2.3.2. Spectrophotometric method (White)
Five grams of honey were dissolved in 25 ml of water,
transferred quantitatively into a 50 ml volumetric flask,
added by 0.5 ml of Carrez solution I and 0.5 ml of
Carrez II and make up to 50 ml with water. The solution
was filtered through paper rejecting the first 10 ml of the
filtrate. Aliquots of 5 ml were put in two test tubes; to
one tube was added 5 ml of distilled water (sample
solution); to the second was added 5 ml of sodium bi-
sulphite solution 0.2% (reference solution). The absor-
bance of the solutions at 284 and 336 nm was
determined using a VARIAN mod. Cary 1E UV–visible.
The quantitative value of HMF was determined both
by the external standard method (p 99% Sigma-Aldrich,
Milan) and by using the proposed formula for the
method reported by IHC (IHC, Stefan Bogdanov, 1999,
pp. 1–54).
2.3.3. Spectrophotometric method (Winkler)
Ten grams of honey were dissolved in 20 ml water
and transferred to a 50 volumetric flask. 2 ml of the
solution and 5.0 ml of p-toluidine solution were put in
two different test tubes; to one tube was added 1 ml of
distilled water (reference solution); to the second, 1 ml of
barbituric acid solution 0.5% (sample solution). The
absorbance of the solutions at 550 nm was determined
using a VARIAN mod. Cary 1E UV–visible. The
quantitative value of HMF was determined both by
the external standard method (p 99%, Sigma-Aldrich,
Milan) and by using the proposed formula for the
method (IHC, Stefan Bogdanov, 1999, pp. 1–54).
2.4. Uncertainty estimation and statistical analyses
The measured uncertainty for HMF analyses in
unifloral honeys was estimated on the basis of the
international laboratory (in-house) according to the
Nordic Committee on Food Analysis (Wood, Nilsson,
& Wallin, 1998) procedure and Eurachem Guide. Re-
Table 2
Relative standard deviation RSDr in HMF determinations by different analytical methods
Analytical methods Average value Acacia
HPLC 0.058 0.054
White 0.060a 0.075
Winkler 0.086 0.104
a
Average RSDr is calculated for each method using all determinations with the exception of Chestnut honey for White method.
275
sults were expressed as expanded/uncertainty (U ) using
2 as coverage factor (95% C.L.), calculated as follows:
U ¼ 2  Cx  RSDr
where 2 is the coverage factor; Cx the concentration of
HMF (mg/kg of honey); RSDr is the relative repeat-
ability standard deviation calculated from duplicate
determination, it was calculated from 40 and 72 repli-
cates for HPLC and spectrophotometric analyses,
respectively. The HPLC replicates were: 8 acacia, 12
citrus, 0 eucalyptus, 4 chestnut and 16 wildflower; the
spectrophotometric replicates were: 12 acacia, 18 citrus,
12 eucalyptus, 6 chestnut and 24 wildflower.
Statgraphics plus software, version 5.0 was used to
perform statistical analyses of the HMF data obtained.
The multiple range tests were performed to evaluate the
statistically significant difference between the HMF
concentration in honeys obtained with three methods.
The model elaborated shows a statistically significant
difference at the 95% confidence level.
3. Results
Table 1 reports the chemical parameters of the anal-
ysed honey: moisture, ash, electrical conductivity, pH,
diastase activity free acids, lactones and total acidity. All
these data are in agreement with those reported in lit-
erature for each unifloral honey (Persano Oddo et al.,
2000).
Table 2 reports the RSDr associated to each analyt-
ical method for HMF determination in each unifloral
honey and an average value for each analytical method
including all honeys samples. Since the calculated RSDr
for chestnut honey by White method was very high
(21.3%) it was not included in the calculation of the
average RSDr. The lowest RSDr in honey analysis was
found with HPLC determination in chestnut honey (3%)
and an average value of 5.8%.
The two spectrophotometric methods show an aver-
age RSDr value of 6.0% and 8.6% using the White and
the Winkler methods, respectively.
Table 3 reports the HMF level in honey samples
analysed by the three methods proposed by the IHC.
Both, for acacia and citrus honeys the highest HMF
values usually were those measured by spectrophoto-
metric analyses, the lowest values were that measured by
Citrus Eucalyptus Chestnut Wildflower
0.059 – 0.030 0.061
0.044 0.060 0.213 0.064
0.082 0.077 0.113 0.077
276 a et al. / Food Control 16 (2005) 273–277
M. Zappal

Table 3
HMF level in commercial monofloral honey determined by HPLC and spectrophotometric methods (HMF ± U *) (mg/kg)
Samples HPLC White Winkler
Ext. st. Formula Ext. st. Formula Ex. St.
Acacia 1 16.2ab ± 1.89 18.4c ± 2.21 20.7d ± 2.50 17.5bc ± 3.03 15.7a ± 2.72
Acacia 2 8.4a ± 0.98 9.1ab ± 1.10 10.0b ± 1.20 11.9c ± 2.06 10.0b ± 1.73
Citrus 1 14.3a ± 1.67 16.4b ± 1.97 18.4c ± 2.21 18.2c ± 3.15 16.7bc ± 2.89
Citrus 2 45.2a ± 5.28 47.0a ± 5.64 54.2b ± 6.50 58.8c ± 10.17 51.5b ± 8.91
Citrus 3 9.4a ± 1.10 9.8a ± 1.18 10.8b ± 1.30 13.5c ± 2.34 11.4b ± 1.97
Citrus 4 8.1a ± 0.95 9.6b ± 1.15 10.3b ± 1.24 11.4c ± 1.97 9.5b ± 1.64
Eucalyptus 1 0.00a 27.7b ± 3.32 31.3c ± 3.76 52.4e ± 9.07 45.4d ± 7.85
Eucalyptus 2 0.00a 6.9b ± 0.83 7.3b ± 0.88 11.5d ± 1.99 9.7c ± 1.68
Chestnut 1 4.1a ± 0.48 4.0a ± 1.72 3.8a ± 1.63 10.4c ± 1.80 8.6b ± 1.49
Chestnut 2 0.00a 0.8b ± 0.34 0.00a 3.2d ± 0.55 2.2c ± 0.38
Wildflower 1 13.7a ± 1.60 14.2a ± 1.70 16.7b ± 2.00 19.1c ± 3.30 16.3b ± 2.82
Wildflower 2 14.2a ± 1.66 13.7a ± 1.64 14.5a ± 1.74 13.9a ± 2.40 11.7b ± 2.02
Wildflower 3 19.6a ± 2.29 17.0b ± 2.04 19.1a ± 2.29 27.1d ± 4.69 23.5c ± 4.07
Wildflower 4 85.5a ± 9.99 83.9a ± 10.1 97.5b ± 11.7 108.8c ± 18.82 99.5b ± 17.2
* U ¼ expanded uncertainty calculated using a cover of factor of 2 (95% confidence level).
a;b;c;d
Means in the same row followed by a different letter are significantly different at 95% C.L.

HPLC. The behaviour of eucalyptus honeys was com-
pletely different from all others (Table 3). Both samples,
when analysed by HPLC, gave no measurable amount
of HMF; the HMF measured by White method, were
27.7 and 31.3 mg/kg of honey for eucalyptus 1, 6.9 and
7.3 mg/kg of honey for eucalyptus 2, using the suggested
formula (IHC, Stefan Bogdanov, 1999, pp. 1–54) and
the external calibration, respectively. The HMF mea-
sured by Winkler method were 52.4 and 45.4 mg/kg of
honey (values out of the legal limit) for eucalyptus 1,
11.5 and 9.7 mg/kg of honey for eucalyptus 2.
At moment it is not possible to explain exactly the
reason of the disagreement among methods, but some
considerations may be done. During the first stages of
heating some HMF precursors are formed: in fact, our
eucalyptus samples in HPLC analyses show another
peak with a maximum absorbance at 256 nm. Previ-
ously, studying the HMF kinetics in unifloral honeys, we
evidentiated a different behaviour of eucalyptus honey
from all other honeys (Fallico et al., 2004). It showed a
longer lag-phase than other honeys. At the beginning of
heating treatment this honey gives the lowest HMF
level, at the end of heating it reaches the same levels
of other honey.
HMF level in chestnut honey was almost the same
both determined by HPLC or White method, it is
heavily over estimated by Winkler method. Although
the first two methods give similar results, the uncertainty
associated with the spectrophotometric determination in
chestnut honey is very high, while RSDr by HPLC was
the lowest value (Table 2).
As chestnut honeys form very low levels of HMF
even after prolonged heating, the authors confirm their
doubts already showed in previous paper (Fallico et al.,
2004), about using HMF level as index of thermal
damage for this unifloral honey.
As concerns the wildflower honeys, the 2nd sample
gave similar HMF values measured by HPLC or spec-
trophotometric methods; in wildflower 1, 3 and 4 the
HMF was the highest when measured by Winkler
method.
Authors confirm suggestions given by International
Commission of Honey (IHC, Stefan Bogdanov, 1999,
pp. 1–54) to not use the Winkler method for determining
HMF in honey, because of carcinogenic of p-toluidine
and of the low precision of this method.
Thus, HPLC method seems to be the more appro-
priate for HMF determination in honey, because the
presence of substances, probably derived by heat or
storage damage, which interfere with the UV methods
did not reveal.
References
Anam, O. O., & Dart, R. K. (1995). Influence of metal ions on
hydroxymethylfurfural formation in honey. Analytical Proceedings
Including Analytical Communications, 32, 515–517.
AOAC (1980). Official Methods of Analysis (13th ed.). Washington,
DC: Association of Official Analytical Chemists, Method 31.111,
p. 521.
Bath, P. K., & Singh, N. (1999). A comparison between Helianthus
annuus & Eucalyptus lanceolatus honey. Food Chemistry, 67, 389–
397.
Belitz, H. D., & Grosch, W. (1999). Food chemistry. Berlin: Springer-
Verlag.
Codex Alimentarius, Alinorm 01/25 (2000). Draft revised standard for
honey at step 8 of the Codex procedure.
EU Directive /110/2001 of 02/12/2001 (L 10/47).
EURACHEM/CITAC Guide (2000). Quantifying uncertainty in
analytical measurement. Ellison, S.R.L., Rosslein, M., & Williams,
A. (Eds.).
Fallico, B., Zappal
a, M., Arena, E., & Verzera, A. (2004). Effects of
heating process on chemical composition and HMF levels in
Sicilian monofloral honeys. Food Chemistry, 85, 305–313.
 a et al. / Food Control 16 (2005) 273–277
M. Zappal
Hase, S., Suzuki, O., Odate, M., & Suzuki, S. (1973). Changes in
quality of honey on heating and storage. I. Changes in hydroxy-
methylfurfural (HMF) content of honey. Journal of Food Science
and Technology, 20, 248–256.
Harmonised methods of the International Honey Commission,
responsible for the methods: Stefan Bogdanov, 1999, pp. 1–54.
Jeuring, J., & Kuppers, F. (1980). High performance liquid chromato-
graphy of furfural and hydroxymethylfurfural in spirits and honey.
Journal of the Association of Official Analytical Chemists, 63, 1215–
1218.
Loveaux, J., Pourtallier, J., & Vorwohl, G. (1973). Methodes d’anal-
yses des miels. Conductivite (Analytical methods for honey
conductivity). Bull. Apic. Inf. Doc. Sci. Tech. Inf., 16, 7.
Mannino, S. (2001). Verification of sample homogenity and calcula-
tion of optimum sample weight. In S. Mannino, & G. Schleining
(Eds.), Practical tasks for food quality assurance (pp. 63). Food
Net-Project No 55792–CP–3–00–1FR–ERASMUS–ETN.
Piazza, M. G., Accorti, M., & Persano Oddo, L. (1991). Electrical
conductivity, ash, colour and specific rotatory power in Italian
unifloral honeys. Apicoltura, 7, 51–63.
Persano Oddo, L., Sabatini, A. G., Accorti, M., Colombo, R.,
Marcazzan, G., Piana, M. L., Piazza, M. G., & Pulcini, P. (2000). I
mieli uniflorali italiani nuove schede di caratterizzazione. Ministero
Politiche Agricole e Forestali, 29–67.
277
Repubblica Italiana: G.U. no. 282, 12/10/1984, Official methods of
honey analysis.
Singh, N., & Bath, P. K. (1997). Quality evaluation of different types of
Indian honey. Food Chemistry, 58, 129–133.
Singh, N., & Bath, P. K. (1998). Relationship between heating &
hydroxymethylfurfural formation in different honey types. Journal
of Food Science and Technology, 35, 154–156.
Singh, N., Singh, S., Bawa, A. S., & Sekhon, K. S. (1988). Honey––its
food uses. Indian Food Packer, 42, 15–25.
White, J. (1979). Spectrophotometric method for hydroxymethyl
furfural in honey. Journal of the Association of Official Analytical
Chemists, 62, 509–514.
Winkler, O. (1955). Beitrag zum Nachwals und zur Bestimmung von
Oxymethylfurfural in Honig und Kunsthonig. Zeitschrift fur
Lebensmittel Untersuchung und Forshung, 102(3), 161–167.
Wood, R., Nilsson, A., & Wallin, H. (1998). Procedure for the
estimation and expression of measurement uncertainty in chemical
analysis developed by the Nordic Committee on Food analysis. In
Quality in the food analysis laboratory (pp. 125–147). UK: The
Royal Society of Chemistry.
Wootton, M., & Ryall, L. (1985). A comparison of Codex Alimen-
tarius Commission and HPLC methods for 5-hydroxymethyl-2-
furaldehyde determination in honey. Journal of Apicoltural
Research, 24(2), 120–124.