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Food Control 16 (2005) 273–277

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Methods for the determination of HMF in honey: a comparison
M. Zappal
a a, B. Fallico
a

a,*

, E. Arena a, A. Verzera

b

Dipartimento di OrtoFloroArboricoltura e Tecnologie Agroalimentari (DOFATA), Facolta di Agraria, Universita di Catania, Via S. Sofia, 98,
Catania 95128, Italy
b
Dipartimento di Chimica Organica e Biologica, Universita di Messina, Papardo, Messina 98168, Italy
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 concentration 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
HMF (5-hydroxymethylfurfuraldehyde) measurement 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 crystallisation or fermentation (Singh, Singh, Bawa, & Sekhon, 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, Zappal
a, 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

pean Union (EU Directive 110/2001) fixed a HMF limit
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 described by White involves measurement of UV absorbance of clarified aqueous honey solutions with and
without bisulphite while that of Winkler involves measurement 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

06 18.120 ± 0.0 ± 0. 1973) by a Delta Ohm HD 8706 conductivity meter.004 0.d.25 17.004 3.51 ± 0.49 13.84 4.29 11. Small differences between the methods resulted only at very low levels.4 ± 0.55 ± 0.929 ± 0.001 n.32 4.12 17.1.03 3.3 ± 0.60 16.17 16. electrical conductivity was measured at 20 C in a 20% (w/v) solution (dry matter basis) in deionised water (Loveaux.0 ± 0. 2.003 ± 0.08 38.84 5.1 ± 1.5 ± 0. 5 lm.35 ± 0.008 0.08 6.2 ± 0.003 0.550 ± 0.8 ± 0.36 29.5 ± 0.36 23.41 38.34 ± 0.51 17. However. RP18.03 3. of no interest for assessing honey quality.0 ± 0.02 7.46 ± 0.35 29.002 0.18 15.002 0.76 ± 0.050 ± 0.1 ± 0.178 ± 0. 282.2 ± 0. citrus.92 ± 0.98 ± 0. 90% water at 1% of acetic acid and 10% methanol.3 ± 0. eucalyptus and chestnut honey and four different samples of citrus and wildflower honey.45 ± 0.14 19.2 ± 0.01 3.4 ± 0.53 24.38 7.01 0.25 24.061 ± 0. Star 330).01 3.23 ± 0. using a kit Phadebas Amylase Test (Pharmacy & Upjohn Diagnostic AB).1.004 0.93 18.0 ± 2. 20 ll.03 3. filtered on 0. the methods yielded comparable values in collaborative studies on three honey samples having different HMF content.84 ± 0. free acids.53 5. respectively (Table 1). fitted with a guard cartridge packed with the same stationary phase (Merck. The wavelength range was 220–660 nm and the chromatograms were monitored at 285 nm. total acidity and pH were measured using a Mettler Toledo MP 220 pH meter according to Official Method (Repubblica Italiana: GU. injection volume.001 1.38 ± 0.5 ± 0.006 0. Zappala et al.3 ± 0.68 ± 0.008 0. Pourtallier.0 ± 0.69 12.63 22. 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.10 15.01 4.03 4.0 ± 0.002 0.91 31.6 ± 0.3 ± 0.7 ml/min.002 1. 1999. 0.53 5.8 ± 0.9 ± 0.46 3. Aim of this research was to compare the HMF level in unifloral honeys measured by the three methods.209 ± 0.04 3.5 ± 0.001 0.9 ± 0. The HPLC column was a Merck Lichrospher.9 ± 0.6 ± 0. Each honey sample was purchased in duplicate in pot of 500 g.49 19. Milan).53 6.8 ± 0.0 ± 0.38 ± 0.5 ± 0.6 ± 0.45 lm filter and immediately injected in a HPLC (Varian 9012Q) equipped with a Diode Array Detector (Varian.66 ± 0. to cover the main range of determination. Stefan Bogdanov. X2 ¼ electrical conductivity in lS/cm at 20 C (Piazza. 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 and the corresponding moisture content (%) was calculated according to AOAC (1980).0 ± 0.08 3. The unifloral honey samples were collected as follow: two different samples of acacia.94 20.004 0.7 ± 0. 2.43 ± 0.002 0.82 4.25 ± 0.70 ± 0.37 ± 0.25 32.6 ± 1.3 ± 0.12 17.2 ± 0. were purchased in some local shopping centres.8 ± 0.04 7.8 ± 0.046 ± 0.7 ± 0.35 26.01 29. The methods were recently tested by the International Honey Commission (IHC.01 16.82 4. Milan).9 ± 0. diastase determinations were conducted by an enzymatic-spectrophotometric method.9 ± 0.01 17.10 0.5 ± 0.6 ± 0.30 10.7 ± 0.001 0.04 43. flow rate.3 ± 0.9 ± 1. Samples Fourteen different honey samples.59 ± 0.94 28.001 0.235 ± 0.002 0. • • • • 2.9 ± 0. pp.8 ± 2. lactones.2 ± 0.3.9 ± 0.27 31.29 18. / Food Control 16 (2005) 273–277 their floral origin or to products that appear during conditioning or storage (Wootton & Ryall.5 ± 0.7 ± 0.1 ± 0.2.84 6.92 5.29 26.16 38.001 0.65 27.11 33.01 16.82 34.001 0.1 ± 0.93 30.688 ± 0.48 13.76 1 2 1 2 3 4 .01 17. eucalyptus. & Vorwohl. 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 conductivity (ms/cm) pH Diastase activity (Schade) Free acids (meq/kg) Lactones (meq/kg) Total acidity (meq/kg) Acacia 1 Acacia 2 Citrus 1 Citrus 2 Citrus 3 Citrus 4 Eucalyptus Eucalyptus Chestnut 1 Chestnut 2 Wildflower Wildflower Wildflower Wildflower 17.6 ± 1.0 ± 0.7 ± 0.10 ± 0.4 ± 0.2 ± 0.22 ± 0.50 34.1 ± 0. Accorti.01 3.02 5.6 ± 0. 125 · 4 mm. 1985). The HPLC conditions were the following: isocratic mobile phase.047 ± 0.31 18.1 ± 0.3.42 14.49 ± 0. chestnut and wildflower as declared in the label of the producer.46 ± 0.1 ± 0.55 ± 0.13 ± 0. 0. & Persano Oddo. HMF determination 2.001 0. 1991).01 27.002 0.35 18.01 3.85 22. 1–54).0 ± 0.4 ± 0.2 ± 0.5 ± 0. All the solvents were HPLC grade (Merck.002 0.01 19. the use of different analytical methods for HMF determination and the use of inaccurate or inadequate procedures are actually a problem. belonging to the following floral origin: acacia. 12/10/1984).23 ± 0.001 0.38 4. HPLC method Five grams of honey samples were diluted up to 50 ml with distilled water.7 ± 0.0 ± 0.274 M.129 ± 0. no.02 3. Material and methods 2.01 15.15 ± 0.29 4.004 1.

060a 0. 2 ml of the solution and 5. Re- Table 2 Relative standard deviation RSDr in HMF determinations by different analytical methods Analytical methods Average value Acacia Citrus Eucalyptus Chestnut Wildflower HPLC White Winkler 0.104 0. Milan) and by using the proposed formula for the method (IHC. respectively.054 0.030 0.213 0. Cary 1E UV–visible. pp.4. Statgraphics plus software. respectively. 12 eucalyptus. 2. diastase activity free acids. to the second. RSDr is the relative repeatability standard deviation calculated from duplicate determination. Cx the concentration of HMF (mg/kg of honey). Zappala et al. Spectrophotometric method (Winkler) Ten grams of honey were dissolved in 20 ml water and transferred to a 50 volumetric flask. added by 0.5 ml of Carrez solution I and 0.0% and 8. The multiple range tests were performed to evaluate the statistically significant difference between the HMF concentration in honeys obtained with three methods. Table 2 reports the RSDr associated to each analytical method for HMF determination in each unifloral honey and an average value for each analytical method including all honeys samples. & Wallin. ash. Sigma-Aldrich. The absorbance of the solutions at 550 nm was determined using a VARIAN mod. Milan) and by using the proposed formula for the method reported by IHC (IHC. The model elaborated shows a statistically significant difference at the 95% confidence level. .058 0. lactones and total acidity.061 0.5 ml of Carrez II and make up to 50 ml with water. Milan).0 was used to perform statistical analyses of the HMF data obtained. 1998) procedure and Eurachem Guide. to one tube was added 5 ml of distilled water (sample solution). 1999. pH.5% (sample solution). Five grams of honey resulted the optimal weight using the Ingamells e Switzer equation (Mannino. 12 citrus. The HPLC replicates were: 8 acacia. the spectrophotometric replicates were: 12 acacia. All these data are in agreement with those reported in literature for each unifloral honey (Persano Oddo et al.3.075 0. Both. calculated as follows: (Sigma-Aldrich. 2001). electrical conductivity. for acacia and citrus honeys the highest HMF values usually were those measured by spectrophotometric analyses. 2000). The quantitative value of HMF was determined both by the external standard method (p 99% Sigma-Aldrich. Stefan Bogdanov.L.2% (reference solution).064 0. to one tube was added 1 ml of distilled water (reference solution).2. 1–54). pp. 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.3%) it was not included in the calculation of the average RSDr. and by comparison the spectrum of HMF standard with that of honey samples.082 – 0.086 0. the lowest values were that measured by 2.6% using the White and the Winkler methods. it was calculated from 40 and 72 replicates for HPLC and spectrophotometric analyses. 4 chestnut and 16 wildflower. 18 citrus.113 0.3.077 a Average RSDr is calculated for each method using all determinations with the exception of Chestnut honey for White method. The two spectrophotometric methods show an average RSDr value of 6. version 5.).059 0.060 0. Cary 1E UV–visible.0 ml of p-toluidine solution were put in two different test tubes. Stefan Bogdanov. Table 3 reports the HMF level in honey samples analysed by the three methods proposed by the IHC. The amount of HMF was determined using an external calibration curve. Nilsson. Results Table 1 reports the chemical parameters of the analysed honey: moisture.M. U ¼ 2  Cx  RSDr where 2 is the coverage factor. 2. measuring the signal at k ¼ 285 nm. 6 chestnut and 24 wildflower. 1 ml of barbituric acid solution 0.8%.3. Since the calculated RSDr for chestnut honey by White method was very high (21.. 1999. Spectrophotometric method (White) Five grams of honey were dissolved in 25 ml of water.077 0. 0 eucalyptus. Aliquots of 5 ml were put in two test tubes. The solution was filtered through paper rejecting the first 10 ml of the filtrate. / Food Control 16 (2005) 273–277 275 sults were expressed as expanded/uncertainty (U ) using 2 as coverage factor (95% C. The lowest RSDr in honey analysis was found with HPLC determination in chestnut honey (3%) and an average value of 5. transferred quantitatively into a 50 ml volumetric flask.044 0. to the second was added 5 ml of sodium bisulphite solution 0. The quantitative value of HMF was determined both by the external standard method (p 99%. The absorbance of the solutions at 284 and 336 nm was determined using a VARIAN mod. 1–54). 3.

66 19. probably derived by heat or storage damage. because of carcinogenic of p-toluidine and of the low precision of this method. K. 67. (Eds. 515–517.20 18. 1999.. it is heavily over estimated by Winkler method.3 mg/kg of honey for eucalyptus 2. (1999).28 9.4c ± 1.L. Stefan Bogdanov.24 31.1a ± 0. Official Methods of Analysis (13th ed. & Williams.4 mg/kg of honey (values out of the legal limit) for eucalyptus 1.R. M.4c ± 2.76 7.4c ± 2. while RSDr by HPLC was the lowest value (Table 2).88 3. Food Chemistry.29 97. st.9c ± 2.). the authors confirm their doubts already showed in previous paper (Fallico et al. Zappala. were 27.5b ± 8.17 13.0a ± 5.3 mg/kg of honey for eucalyptus 1. Food chemistry. O. / Food Control 16 (2005) 273–277 Table 3 HMF level in commercial monofloral honey determined by HPLC and spectrophotometric methods (HMF ± U *) (mg/kg) Samples Acacia 1 Acacia 2 Citrus 1 Citrus 2 Citrus 3 Citrus 4 Eucalyptus Eucalyptus Chestnut 1 Chestnut 2 Wildflower Wildflower Wildflower Wildflower 1 2 1 2 3 4 HPLC White Winkler Ext.3b ± 1.03 11.3c ± 3.5bc ± 3. Codex Alimentarius. S.4a ± 0.2a ± 1. st.02 23.00a 16..5b ± 17.4b ± 1.82 11.5c ± 4. M.06 18. about using HMF level as index of thermal damage for this unifloral honey.97 47.9a ± 2.69 108. Alinorm 01/25 (2000).7 17. Formula Ext. At the beginning of heating treatment this honey gives the lowest HMF level. .00a 13.7 and 31.5b ± 11.5c ± 2.07 11. (1995).68 8. 16.63 0. D.6b ± 1. DC: Association of Official Analytical Chemists. EURACHEM/CITAC Guide (2000). Although the first two methods give similar results.15 27.97 9.74 19.c.00a 4. During the first stages of heating some HMF precursors are formed: in fact. Zappala et al. Belitz.8a ± 1. pp.2d ± 0. we evidentiated a different behaviour of eucalyptus honey from all other honeys (Fallico et al. 389– 397.2 * U ¼ expanded uncertainty calculated using a cover of factor of 2 (95% confidence level).5d ± 1. Berlin: SpringerVerlag.7b ± 2. O. 3 and 4 the HMF was the highest when measured by Winkler method.1d ± 4. pp.2c ± 3.5a ± 1.99 18.98 14. AOAC (1980). at the end of heating it reaches the same levels of other honey.67 45.2ab ± 1.0b ± 1.55 19.7 mg/kg of honey for eucalyptus 2.73 16. Fallico.0a ± 1.7a ± 1.4a ± 1.2b ± 6. p.3b ± 2. W.30 10.50 10. At moment it is not possible to explain exactly the reason of the disagreement among methods. Draft revised standard for honey at step 8 of the Codex procedure.83 4.15 58.38 16.85 9.95 0. in wildflower 1. 6. Thus.6a ± 2.9b ± 0.L. respectively. Rosslein.1 20.4d ± 7. Influence of metal ions on hydroxymethylfurfural formation in honey. Formula Ex.2a ± 5.89 51.34 14. The HMF measured by Winkler method were 52.b.. Effects of heating process on chemical composition and HMF levels in Sicilian monofloral honeys. & Grosch. Arena.8c ± 10. References Anam.21 9. EU Directive /110/2001 of 02/12/2001 (L 10/47).1c ± 3. Analytical Proceedings Including Analytical Communications.7d ± 2.49 2...4 and 45. our eucalyptus samples in HPLC analyses show another peak with a maximum absorbance at 256 nm.10 8.8b ± 0. HPLC method seems to be the more appropriate for HMF determination in honey. 1–54) and the external calibration. the uncertainty associated with the spectrophotometric determination in chestnut honey is very high. A. (2004).99 10.60 14.d Means in the same row followed by a different letter are significantly different at 95% C.). A. B.32 6.4b ± 1. Method 31.40 27. but some considerations may be done. St.3b ± 0.. 85. Authors confirm suggestions given by International Commission of Honey (IHC..48 0.80 3. Ellison. E. 305–313. a.0b ± 1.64 17. studying the HMF kinetics in unifloral honeys. A comparison between Helianthus annuus & Eucalyptus lanceolatus honey.7b ± 2. & Verzera.4e ± 9.7b ± 3.6b ± 1..18 9.97 52.111. the 2nd sample gave similar HMF values measured by HPLC or spectrophotometric methods.50 10. which interfere with the UV methods did not reveal.89 8. 1–54) to not use the Winkler method for determining HMF in honey. & Dart. H.1ab ± 1. when analysed by HPLC. 11. 1999. 521.64 9. As concerns the wildflower honeys. R.2c ± 0.8a ± 1. the HMF measured by White method.00a 0. because the presence of substances.1a ± 2.5 and 9. K.7a ± 1. & Singh. Previously.9a ± 10. Washington.72 0.07 99.8b ± 1.8c ± 18. It showed a longer lag-phase than other honeys.04 83.30 13.5a ± 9. P.9 and 7.276 M. gave no measurable amount of HMF. HPLC. N.82 15. Both samples.3a ± 1. As chestnut honeys form very low levels of HMF even after prolonged heating.10 16. Stefan Bogdanov. Bath.64 45. Quantifying uncertainty in analytical measurement.7c ± 1. The behaviour of eucalyptus honeys was completely different from all others (Table 3).21 54.34 11.7bc ± 2.7a ± 2.29 85..4c ± 1.2a ± 1.1a ± 0. 32.72 10. 2004). HMF level in chestnut honey was almost the same both determined by HPLC or White method.0b ± 2.00 14.91 11. Food Chemistry.5b ± 1. 2004). (1999). using the suggested formula (IHC.70 13..

Piazza.U.. 1215– 1218. (1988). N. Accorti. Procedure for the estimation and expression of measurement uncertainty in chemical analysis developed by the Nordic Committee on Food analysis. Mannino. R. 248–256. 20. M. ash. Tech. Apic. 154–156. 125–147). M. Apicoltura. (1979). Journal of the Association of Official Analytical Chemists. Suzuki.. (2001). Mannino.. & Pulcini. (1997). J. M. White. 62.. Colombo. & Suzuki. & G. Indian Food Packer.. Food Chemistry. G. & Bath.. / Food Control 16 (2005) 273–277 Hase. O.). & Kuppers. M. Inf. J.. Singh. Conductivite (Analytical methods for honey conductivity). 282. Spectrophotometric method for hydroxymethyl furfural in honey. Singh. S. Singh. L. & Bath. & Sekhon. Piana. K... Changes in quality of honey on heating and storage. Pourtallier. Winkler. Journal of Food Science and Technology. Odate. M. (1973). I. S... Official methods of honey analysis. 51–63. 102(3). Journal of Apicoltural Research.. J. Sci. Zappala et al. A. N. M. K... & Persano Oddo. Loveaux. (1955). S. 15–25. I mieli uniflorali italiani nuove schede di caratterizzazione. Electrical conductivity. 63). 35. Nilsson. & Ryall. F. UK: The Royal Society of Chemistry. G. High performance liquid chromatography of furfural and hydroxymethylfurfural in spirits and honey. (1973). 129–133. Doc. Bull. pp. L. 42. 120–124. L. no. 7.. R. J. Verification of sample homogenity and calculation of optimum sample weight. colour and specific rotatory power in Italian unifloral honeys. H. L. 63. (1998). & Wallin. Bawa. P. responsible for the methods: Stefan Bogdanov. S. Changes in hydroxymethylfurfural (HMF) content of honey. & Vorwohl. A. O. (1985). (1991). K. P.. Singh. In Quality in the food analysis laboratory (pp. G. 1999.. 161–167. 29–67. (1980). Honey––its food uses. Journal of Food Science and Technology.. Practical tasks for food quality assurance (pp. 12/10/1984. S.. Food Net-Project No 55792–CP–3–00–1FR–ERASMUS–ETN. Ministero Politiche Agricole e Forestali. G. Wood. Marcazzan. P.M. 1–54. Accorti. Relationship between heating & hydroxymethylfurfural formation in different honey types. 58. Beitrag zum Nachwals und zur Bestimmung von Oxymethylfurfural in Honig und Kunsthonig. Wootton.. M. Sabatini.. A comparison of Codex Alimentarius Commission and HPLC methods for 5-hydroxymethyl-2furaldehyde determination in honey. S. Schleining (Eds.. 16. Zeitschrift fur Lebensmittel Untersuchung und Forshung. 509–514.. Methodes d’analyses des miels. G. 277 Repubblica Italiana: G. In S. 24(2). (2000). Piazza. Jeuring. Quality evaluation of different types of Indian honey. Journal of the Association of Official Analytical Chemists.. Inf. . (1998). 7. Harmonised methods of the International Honey Commission. A. N. Persano Oddo.