Food Control 16 (2005) 273–277 www.elsevier.com/locate/foodcont

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: [email protected] (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

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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 inadequate procedures are actually a problem. The methods were recently tested by the International Honey Commission (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 Fourteen different honey samples, belonging to the following floral origin: acacia, citrus, eucalyptus, chestnut and wildflower as declared in the label of the producer, 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 wildflower 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

and the corresponding moisture content (%) was calculated 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 measured 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 enzymatic-spectrophotometric method, using a kit Phadebas Amylase Test (Pharmacy & Upjohn Diagnostic AB).

2.3. HMF determination 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, RP18, 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 chromatograms 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 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

17.4 ± 0.01 17.0 ± 0.01 16.6 ± 0.01 17.2 ± 0.01 19.1 ± 0.14 19.5 ± 0.01 15.5 ± 0.17 16.5 ± 0.35 18.0 ± 0.12 17.9 ± 0.12 17.1 ± 0.10 15.8 ± 0.01 16.9 ± 0.06 18.0 ± 0.10

0.003 ± 0.001 n.d. 0.046 ± 0.002 0.120 ± 0.001 0.047 ± 0.001 0.050 ± 0.004 0.209 ± 0.002 0.235 ± 0.001 0.688 ± 0.002 0.929 ± 0.006 0.129 ± 0.001 0.061 ± 0.01 0.550 ± 0.004 0.178 ± 0.002

0.15 ± 0.001 0.13 ± 0.002 0.22 ± 0.003 0.35 ± 0.001 0.23 ± 0.002 0.23 ± 0.008 0.51 ± 0.004 0.55 ± 0.002 1.34 ± 0.004 1.59 ± 0.002 0.37 ± 0.001 0.25 ± 0.001 1.10 ± 0.008 0.45 ± 0.004

3.38 ± 0.02 3.55 ± 0.03 3.46 ± 0.01 3.46 ± 0.01 3.49 ± 0.03 3.43 ± 0.01 3.68 ± 0.01 3.66 ± 0.01 4.98 ± 0.02 5.84 ± 0.04 3.92 ± 0.08 3.70 ± 0.03 4.38 ± 0.03 3.76 ± 0.04

7.7 ± 0.42 14.8 ± 0.38 7.8 ± 0.02 7.9 ± 0.30 10.0 ± 0.69 12.0 ± 0.93 18.2 ± 0.65 27.9 ± 1.18 15.7 ± 0.63 22.2 ± 0.94 20.0 ± 0.31 18.6 ± 0.29 18.9 ± 0.49 13.7 ± 0.49

19.9 ± 0.48 13.3 ± 0.29 26.3 ± 0.35 29.8 ± 0.35 26.0 ± 0.01 27.0 ± 0.01 29.3 ± 0.50 34.1 ± 0.25 17.3 ± 0.29 11.4 ± 0.25 24.1 ± 0.85 22.9 ± 0.25 32.5 ± 0.41 38.3 ± 0.29

4.2 ± 0.38 4.3 ± 0.82 4.2 ± 0.46 3.9 ± 0.92 5.0 ± 0.32 4.7 ± 0.82 4.8 ± 0.84 4.5 ± 0.53 6.5 ± 0.53 5.2 ± 0.53 5.5 ± 0.84 6.0 ± 2.08 6.1 ± 0.84 5.2 ± 0.53

24.1 ± 0.51 17.6 ± 0.93 30.4 ± 0.11 33.6 ± 1.27 31.0 ± 0.91 31.7 ± 0.82 34.1 ± 1.08 38.6 ± 0.36 23.7 ± 0.60 16.5 ± 0.36 29.6 ± 0.94 28.8 ± 2.16 38.6 ± 1.04 43.4 ± 0.76

1 2

1 2 3 4

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sults were expressed as expanded/uncertainty (U ) using 2 as coverage factor (95% C.L.), calculated as follows:

(Sigma-Aldrich, Milan), and by comparison the spectrum 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).

U ¼ 2  Cx  RSDr where 2 is the coverage factor; Cx the concentration of HMF (mg/kg of honey); RSDr is the relative repeatability standard deviation calculated from duplicate determination, it was calculated from 40 and 72 replicates 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.

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 bisulphite solution 0.2% (reference solution). The absorbance 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).

3. Results Table 1 reports the chemical parameters of the analysed honey: moisture, ash, electrical conductivity, pH, diastase activity free acids, lactones and total acidity. All these data are in agreement with those reported in literature for each unifloral honey (Persano Oddo et al., 2000). 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. 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 average 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 spectrophotometric analyses, the lowest values were that measured by

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

Citrus

Eucalyptus

Chestnut

Wildflower

HPLC White Winkler

0.058 0.060a 0.086

0.054 0.075 0.104

0.059 0.044 0.082

– 0.060 0.077

0.030 0.213 0.113

0.061 0.064 0.077

a

Average RSDr is calculated for each method using all determinations with the exception of Chestnut honey for White method.

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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. st.

Formula

Ext. st.

Formula

Ex. St.

16.2ab ± 1.89 8.4a ± 0.98 14.3a ± 1.67 45.2a ± 5.28 9.4a ± 1.10 8.1a ± 0.95 0.00a 0.00a 4.1a ± 0.48 0.00a 13.7a ± 1.60 14.2a ± 1.66 19.6a ± 2.29 85.5a ± 9.99

18.4c ± 2.21 9.1ab ± 1.10 16.4b ± 1.97 47.0a ± 5.64 9.8a ± 1.18 9.6b ± 1.15 27.7b ± 3.32 6.9b ± 0.83 4.0a ± 1.72 0.8b ± 0.34 14.2a ± 1.70 13.7a ± 1.64 17.0b ± 2.04 83.9a ± 10.1

20.7d ± 2.50 10.0b ± 1.20 18.4c ± 2.21 54.2b ± 6.50 10.8b ± 1.30 10.3b ± 1.24 31.3c ± 3.76 7.3b ± 0.88 3.8a ± 1.63 0.00a 16.7b ± 2.00 14.5a ± 1.74 19.1a ± 2.29 97.5b ± 11.7

17.5bc ± 3.03 11.9c ± 2.06 18.2c ± 3.15 58.8c ± 10.17 13.5c ± 2.34 11.4c ± 1.97 52.4e ± 9.07 11.5d ± 1.99 10.4c ± 1.80 3.2d ± 0.55 19.1c ± 3.30 13.9a ± 2.40 27.1d ± 4.69 108.8c ± 18.82

15.7a ± 2.72 10.0b ± 1.73 16.7bc ± 2.89 51.5b ± 8.91 11.4b ± 1.97 9.5b ± 1.64 45.4d ± 7.85 9.7c ± 1.68 8.6b ± 1.49 2.2c ± 0.38 16.3b ± 2.82 11.7b ± 2.02 23.5c ± 4.07 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 completely 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 measured 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. Previously, 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 spectrophotometric 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 appropriate 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.

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