Food and Chemical Toxicology 48 (2010) 23882392

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Food and Chemical Toxicology

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High 5-hydroxymethylfurfural concentrations are found in Malaysian honey samples stored for more than one year
M.I. Khalil, S.A. Sulaiman *, S.H. Gan
Department of Pharmacology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia

a r t i c l e

i n f o

a b s t r a c t
5-Hydroxymethylfurfural (HMF) content is an indicator of the purity of honey. High concentrations of HMF in honey indicate overheating, poor storage conditions and old honey. This study investigated the HMF content of nine Malaysian honey samples, as well as the correlation of HMF formation with physicochemical properties of honey. Based on the recommendation by the International Honey Commission, three methods for the determination of HMF were used: (1) high performance liquid chromatography (HPLC), (2) White spectrophotometry and (3) Winkler spectrophotometry methods. HPLC and White spectrophotometric results yielded almost similar values, whereas the Winkler method showed higher readings. The physicochemical properties of honey (pH, free acids, lactones and total acids) showed signicant correlation with HMF content and may provide parameters that could be used to make quick assessments of honey quality. The HMF content of fresh Malaysian honey samples stored for 36 months (at 2.8024.87 mg/kg) was within the internationally recommended value (80 mg/kg for tropical honeys), while honey samples stored for longer periods (1224 months) contained much higher HMF concentrations (128.191131.76 mg/kg). Therefore, it is recommended that honey should generally be consumed within one year, regardless of the type. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 22 March 2010 Accepted 27 May 2010

Keywords: Malaysian honey 5-Hydroxymethylfurfural Purity Physicochemical properties

1. Introduction Honey is a complex mixture of water, sugars (glucose, fructose, sucrose, maltose and higher sugars), gluconic acid, lactone, nitrogenous compounds, minerals and some vitamins (Ramirez et al., 2000). Hydroxymethylfurfural (HMF) is a cyclic aldehyde produced as a result of sugar degradation (Ramirez et al., 2000). It is said that the presence of simple sugars (glucose and fructose) and many acids in honey is a favorable condition for the production of this substance. It has been reported that HMF and its congener compounds are spontaneously formed in carbohydrate-containing foods by Maillard reactions (the non-enzymatic browning reaction) or by an acid-catalyzed dehydration of hexoses (Belitz and Grosch, 1999). HMF is usually absent in fresh and untreated foods (Askar, 1984), but its concentration is also reported to increase as a result of heating processes (Bath and Singh, 1999; Fallico et al., 2004) or due to long-term storage. For this reason, HMF is a recognized parameter related to the freshness and quality of such foods. Several factors inuence the formation of HMF in honey during storage conditions. These factors include the following: (1) the use of metallic containers (White, 1979) and (2) the physicochemical
* Corresponding author. Tel.: +60 9 7676124; fax: +60 9 7653370. E-mail address: sbsamrah@kb.usm.my (S.A. Sulaiman). 0278-6915/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.fct.2010.05.076

properties (the pH, total acidity, mineral content) of honey itself, which are related to the oral source from which the honey has been extracted (Anam and Dart, 1995), the humidity and from thermal and/or photochemical stress (Spano et al., 2006). The Codex Alimentarius (Alinorm 01/25 2000) has established that the HMF content of honey after processing and/or blending must not be higher than 80 mg/kg. The European Union (EU Directive 110/2001), however, recommends a lower limit of 40 mg/kg with the following exceptions: 80 mg/kg is allowed for honey that originates from countries or regions with tropical temperatures, while a lower limit at only 15 mg/kg is allowed for honey with low enzymatic levels. The International Honey Commission (1999) recommends three methods for the determination of HMF. These methods include two spectrophotometric methods widely used in routine analysis, determination based on protocols by Winkler (1955) and White (1979), as well as a chromatographic technique using high performance liquid chromatography (HPLC). However, to date, there is no available literature that compares the accuracies of the three methods or reports the correlation between physicochemical properties of honey and their HMF levels. This is important because high HMF concentrations have reportedly caused mutagenic activities that can be dangerous for humans if consumed (Surh et al., 1994; Kubi and Ingr, 1998; Janzowski et al., 2000). A correlation between physicochemical properties of honey and their HMF levels

M.I. Khalil et al. / Food and Chemical Toxicology 48 (2010) 23882392 Table 1 The investigated honey samples with their different storage conditions and treatments. Sample 1 2 3 4 5 6 7 8 9 10 Tualang honey 1* Tualang honey 2* Tualang honey 3^ Tualang honey 4^ Tualang honey 5 Gelam honey 6# Gelam honey 7# Borneo tropical honey Honey B Manuka honey Stored condition Evaporated and non-radiated Evaporated and radiated Non-evaporated and non-radiated Evaporated but non-radiated Evaporated and non-radiated Non-evaporated and non-radiated Evaporated and non-radiated Non-evaporated, heated to 50 C Malaysian honey randomly purchased from a supermarket Used as a gold standard for comparison

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Average storage time (months) 24 24 6 6 12 6 6 6 12 12

N/B: The pairs of honey samples 1* and 2*, 3^ and 4^ & 6# and 7# are from the same source but were either treated or stored differently.

(if established) will be a quick method that can safeguard humans from consuming honey samples with high HMF contents because it is easier and quicker to establish the physicochemical properties of honey samples in the laboratory using simple tests than to measure HMF levels. The aims of this study were to (i) analyze the HMF content in nine Malaysian honey samples stored and treated under various conditions; (ii) compare their HMF levels determined by the three methods; (iii) investigate the effects of storage duration on HMF formation; and (iv) correlate other physicochemical properties of honey (such as the pH, free acids, lactones, total acids and moisture content) with HMF formation.
2. Materials and methods 2.1. Honey samples Nine honey samples from Malaysia that have been stored or treated differently were used in this investigation. They were Tualang honey (n = 5), Gelam honey (n = 2), Borneo tropical honey (n = 1) and a random Malaysian honey sample bought of the supermarket shelf named Honey B (n = 1; Table 1). Since Manuka honey has been extensively investigated, it was used as the gold standard for comparison. In this study, we have used Manuka Honey Active 5+, Comvita, from New Zealand. All samples were stored at room temperature (2530 C) before analysis. 2.2. Physicochemical properties of honey 2.2.1. Free acids, pH, lactones and total acidity The pH of the honey solution containing 10 g of each honey diluted with distilled water (75 ml) was measured by using a pH meter (Accumet Basic AB15, Fisher Scientic Co., USA) (AOAC, 1990). To obtain the free acids, each sample was titrated with sodium hydroxide added in excess to hydrolyze any lactose present, followed by an immediate back-titration with hydrochloric acid. The total acidity (in meq/kg) was calculated as free acidity plus lactone. To measure the total acidity, 10 g of honey sample was dissolved in 75 ml of carbon dioxide-free water in a 250 ml beaker followed by stirring with a magnetic stirrer. The electrodes of the pH meter were immersed into the solution for pH recording. Then, the honey solution was titrated with 0.05 N NaOH at a rate of 5 ml/min until pH 8.5 was achieved. The burette reading was used to titrate the exact amount of NaOH used. After that, 10 ml of 0.05 N NaOH was added immediately by pipette, followed by an immediate back-titration with 0.05 N HCl from a 10 ml burette until the pH reached 8.3. The procedure was also repeated with a reagent blank as a negative control. The results were expressed as meq/kg (AOAC, 1990). 2.2.2. Moisture content The conventional-drying oven method was carried out as described in the Association of Ofcial Analytical Chemists (AOAC) method number 925.45 (AOAC, 1990). The moisture content was determined by drying a weighed amount of the sample at 105 C for 3 h (or until a constant weight was obtained). Samples were analyzed in triplicate, and the corresponding moisture content was calculated as a percentage. 2.3. Determination of HMF content

Carrez solution II [consisting of 30 g of zinc acetate, Zn(CH3 COO)22H2O in 100 ml of water] were added, and the mixture was made up to 50 ml with water. The solution was ltered using lter paper after rejecting the rst 10 ml of the ltrate. Then, aliquots of 5 ml were transferred into two test tubes each. To the rst tube, 5 ml of distilled water (sample solution) was added, while 5 ml of sodium bisulphate solution 0.2% (reference solution) was added to the second tube. The absorbance of the solutions at 284 and 336 nm was determined using a T 80 UV/ VIS spectrophotometer (ChromoTek GmbH, Germany). The quantitative value of HMF was determined both by the external standard method (SigmaAldrich, Saint Louis, MO, USA) and by using the proposed formula for the method reported by International Honey Commission (1999).

2.3.2. The Winkler spectrophotometric method (Winkler, 1955) The honey samples (10 g each) were dissolved in 20 ml water and transferred to a 50 ml volumetric ask. Next, 2 ml of the solution and 5.0 ml of p-toluidine solution were put into two different test tubes; 1 ml of distilled water (reference solution) was added into one tube; while to the second tube, 1 ml of barbituric acid solution 0.5% (sample solution) was added. The absorbance of the solutions at 550 nm was determined using a T 80 UV/VIS spectrophotometer (ChromoTek GmbH, Germany). The quantitative value of HMF was determined both by an external standard method (SigmaAldrich) and by using the proposed formula for the method (International Honey Commission, 1999).

2.3.3. HPLC method The HPLC method used was based on the method published by the International Honey Commission in 1999. Briey, the honey samples (10 g each) were diluted to 50 ml with distilled water, ltered using a 0.45 lm nylon membrane lter and injected (20 ll) into an HPLC system (Waters 2695, Milford, MA, USA) equipped with a Photodiode Array Detector (Waters 2996). The HPLC column was a Merck Purospher Star, RP-18e, (125 4 mm, 5 lm) tted with a guard cartridge packed with the same stationary phase (Merck, Germany). The HPLC method included an isocratic mobile phase, 90% water and 10% methanol with a ow rate of 1.0 ml/min. All solvents used were of HPLC grade. The detection wavelength was 200450 nm with specic monitoring at 285 nm. The HMF content of the sample was calculated by comparing the corresponding peak areas of the sample and those of the standard solutions of HMF (SigmaAldrich, USA) after correcting for the honey dilution. There was a linear relationship (R2 = 0.9997; Fig. 1) between the concentration and the area of the HMF peak (results are expressed in mg/kg).

1,800,000 1,600,000 1,400,000

y = 7E+06x + 97011 2 R = 0.9997

Area of HMF peak

1,200,000 1,000,000 800,000 600,000 400,000 200,000 0 0

0.05 0.1 0.15 0.2 0.25

2.3.1. The White spectrophotometric method (White, 1979) The honey samples (5 g each) were dissolved in 25 ml of water and transferred into a 50 ml volumetric ask. Then, 0.5 ml of Carrez solution I [consisting of 15 g of potassium hexacyanoferrate(II), K4Fe(CN)63H2O in 100 ml of water] and 0.5 ml of

HMF concentrartion (g/ml)

Fig. 1. Linear relationship between the concentration of HMF and peak area.

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Table 2 Physicochemical properties of nine Malaysian honey samples (mean SD; n = 3) in comparison to Manuka Honey. Samples Tualang honey 1 Tualang honey 2 Tualang honey 3 Tualang honey 4 Tualang honey 5 Gelam honey 6 Gelam honey 7 Borneo tropical honey Honey B Mean Manuka honey Moisture (%) 18.78 0.65 18.60 0.57 22.32 0.32 17.38 0.44 16.39 0.45 20.33 0.49 18.51 0.52 14.46 0.48 12.71 0.45 17.72 2.75 9.28 0.37 pH 3.44 0.04 3.48 0.03 3.67 0.02 3.69 0.06 3.62 0.10 3.61 0.06 3.55 0.03 3.89 0.04 3.61 0.09 3.62 0.12 3.99 0.02 Free acids (meq/kg) 81.83 2.02 76.50 2.50 37.33 3.82 38.00 1.80 64.33 3.82 50.93 3.82 37.50 2.50 39.50 1.32 29.33 1.44 50.59 18.00 34.00 2.50 Lactones (meq/kg) 4.25 0.43 5.75 1.15 7.58 0.80 8.00 0.87 4.15 0.56 8.82 0.28 9.00 0.87 8.33 0.58 5.33 0.76 6.80 1.83 8.67 0.58 Total acidity (meq/kg) 86.08 2.38 82.25 3.63 44.92 3.17 46.00 1.00 68.48 3.28 59.75 3.55 46.50 3.28 47.83 1.89 34.67 1.76 57.39 16.94 42.67 3.01

2.4. Statistical analysis Data were analyzed using the SPSS software (Statistical Packages for Social Science 12.0 (SPSS Inc., USA). A one-way analysis of variance (ANOVA), followed by Tukeys honestly signicant difference post hoc test with a = 0.05, was used to compare the differences between the honey samples. Correlations were determined by a regression curve t model where HMF was the independent variable and pH, free acids, lactone and total acids were the dependent variables. The assays were carried out in triplicate, and the results are expressed as mean values standard deviations (SDs).

3. Results Table 2 lists the physicochemical characteristics of the nine investigated honey samples.
1600
a

1400 1200 1000 800 600 400

a a b a HPLC White method Winkler method a

The moisture content of the Malaysian honey samples ranged between 14.46% and 22.32%. Tualang honey 3, which was considered fresh (stored for six months), contained the highest percentage of moisture (22.32%), and Manuka honey reported the lowest (9.28%) levels. Among the nine Malaysian honey samples, Tualang honey 1 (evaporated, non-radiated and stored for 24 months duration) had the lowest pH (3.44) and contained the highest free and total acids (81.83 and 86.06 meq/kg, respectively). The Borneo tropical honey reported the highest pH value (3.89). The spectrophotometric method described by Winkler (1955) showed higher HMF readings while the spectrophotometric method described by White (1979) and the HPLC method tended to yield similar but lower readings (Fig. 2). Based on the HPLC method (Table 3), the HMF concentrations in ve honey samples (Tualang honey samples 3 and 4, Gelam honey samples 1 and 2, and Borneo tropical honey) stored over a shorter duration (up to six months) tended to be lower (2.80 24.87 mg/kg). However, Tualang honey samples 1 and 2 stored for 24 months had the highest HMF values (1131.76 and 986.57 mg/kg, respectively). A similar trend was observed for the HMF concentrations determined with the spectrophotometric methods. Table 4 shows that there was a very strong correlation (R2 = 0.912) between storage duration and HMF concentrations. Strong correlations were also observed between free or total acids of honey samples and HMF concentrations (R2 = 0.786 and 0.763, respectively), while HMF content moderately correlated with pH and lactone concentrations.

200 0

ab

4. Discussion
6 12 24

-200
Fig. 2. The effect of storage duration on mean HMF levels determined by the three methods recommended by International Honey Commission (1999). Values (bars) are mean SD and different letters indicate signicant differences (p < 0.05).

The water or moisture content in honey generally depends on the botanical origin of the sample, the processing techniques and the storage conditions (Conti, 2000). Moisture present in a honey sample is also related to climatic conditions and the degree of maturity of the honey, while any anomalous value may be an

Table 3 HMF levels of Malaysian honey samples determined by HPLC and spectrophotometric methods (mean SD; n = 3). Sample/method Tualang honey 1 Tualang honey 2 Tualang honey 3 Tualang honey 4 Tualang honey 5 Gelam honey 1 Gelam honey 2 Borneo tropical honey Honey B Manuka honey HPLC 1131.76 18.61a 986.57 9.81b 4.19 0.45f 2.86 0.15f 206.06 5.51c 6.65 1.47ef 5.97 0.29ef 11.83 0.70ef 128.19 9.18d 26.75 1.90e White, 1979 1202.43 25.40a 975.57 13.19b 7.86 1.58e 4.19 0.73e 228.72 17.20c 7.31 0.90e 7.63 0.74e 13.16 2.16e 129.52 5.13d 25.41 3.01e Winkler, 1955 1344.76 31.57a 1043.57 46.67b 12.19 0.45e 8.19 1.23e 383.39 5.42c 16.65 1.38e 11.97 1.24e 21.16 2.16e 181.52 13.93d 42.41 3.90e Mean values of the three methods 1226.32 108.49a 1001.91 36.50b 8.08 4.00e 5.08 2.78e 272.72 96.51c 10.20 5.59e 8.52 3.10e 15.38 5.05de 146.41 30.41cd 31.53 9.45de

Note: In each column, values with different letters (superscripts) indicate signicant differences (p < 0.05).

M.I. Khalil et al. / Food and Chemical Toxicology 48 (2010) 23882392 Table 4 Correlation established between HMF and the physicochemical parameters. Chemical parameter Storage time pH Free acid Lactone Total acidity Equation Y = 0.0153x + 7.0782 Y = 0.0002x + 3.7194 Y = 0.0361x + 39.6167 Y = 0.0027x + 7.5587 Y = 0.0334x + 47.1777 R2 0.912 0.407 0.786 0.411 0.763 F 93.16 6.17 33.11 6.28 29.04 p <0.001 0.035 <0.001 0.033 <0.001

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indicator of contaminants or poor honey quality. This is because any yeast cells present in honey will be able to ferment the sugar if water is present in high amounts. Our results indicated that only two honey samples (Tualang honey 3 and Gelam honey 6) that were not subjected to evaporation, showed levels of HMF that were higher than the 20% limit permitted by the Council Directive of the European Union (2002). This conrmed that, in general, the moisture content in Malaysian honey samples previously treated by evaporation produced low but acceptable fermentation rates similar to those previously reported by Tumin et al. (2005) for ve types of Malaysian honey. Therefore, evaporation is an important step in maintaining the freshness of honey. The pH is a useful indicator for possible microbial contamination (Conti, 2000). Additionally, pH is an important factor during the extraction and storage of honey because it is related to the stability and the shelf life of the product (Terrab et al., 2004). As previously reported (Conti et al., 1998), most bacteria and molds grow in neutral and mildly alkaline environments, respectively. In contrast, yeasts require an acidic environment (pH range of 4.04.5) and do not grow in alkaline media. Since the mean pH of the nine Malaysian honey samples analyzed was 3.62 (range 3.443.89), which was much lower than that of Manuka honey (3.99), it is possible that Malaysian honey samples may have superior antibacterial properties compared to Manuka honey; this nding was also reported by Tan et al. (2009). The free acid content of each of the six honey samples was within the permitted range proposed by Codex Alimentarius (2000) (<50 meq/kg). The other three samples (Tualang honey samples 1 and 2, which were stored for 24 months), as well as the Tualang honey 5 sample (non-radiated and stored for 12 months) exceeded the permitted values. High levels of free acids may indicate that sugars are present in samples that have undergone fermentation in the presence of yeasts. It is well known that during fermentation, glucose and fructose are converted into carbon dioxide and alcohol. In the presence of oxygen, the alcohol is further hydrolyzed and converted to acetic acid, which contributes to the free acid content in the honey. Lactones are another contributor to the total acidity in honey where it is present at levels between 4.15 and 9.00 meq/kg in the samples. The average total acidity of the Malaysian honey samples ranged between 34.67 and 86.08 meq/kg. Tualang honey samples 1 and 2, which were stored for 24 months, showed the highest readings (86.08 and 82.25 meq/kg, respectively) compared to other samples including Manuka honey (42.67 meq/kg). The total acid content also includes organic acids, particularly gluconic acid, in equilibrium with lactones or esters and inorganic ions, such as phosphates and chlorides. Besides the fact that total acidity is inuenced by variations in harvest seasons (Singh and Bath, 1996), our results illustrated the possible inuence of oral type and storage duration on the total acidity of honey samples. In terms of HMF content, according to the International Commission of Honey (International Honey Commission, 1999), the Winkler (1955) spectrophotometric method is not the preferred method for determining the HMF content in honey samples due to the carcinogenicity of the p-toluidine required by the method, as well as its reported lower accuracy. We tend to agree with this

recommendation as the presence of artifacts, derived by heat or storage damage, may interfere with the spectrophotometric methods and may not be revealed when HPLC is used. However, the spectrophotometric method established by White (1979) still yielded HMF concentrations comparable to those produced with HPLC. Based on the HPLC method, HMF concentrations of ve of the honey samples (Tualang honey samples 3 and 4, Gelam honey samples 1 and 2, as well as Borneo tropical honey) stored for up to six months were found to range from 2.80 to 24.87 mg/kg. This is within the allowed maximum limit of 40 mg/kg as recommended by the Turkish Alimentarus Codex (Anonymous, 2003) for honey samples. These results are in contradiction to those reported by Lagrange and Sanders (1988), which stated that honey samples produced from countries with subtropical climates have high HMF concentrations that generally exceeded 40 mg/kg. However, Tualang honey samples 1 and 2 stored for 24 months recorded the highest HMF readings (1131.76 and 986.57 mg/kg, respectively). Tualang honey 5 and Honey Bwith storage durations of 12 monthsalso yielded high HMF values (206.06 and 128.19 mg/kg, respectively) when compared to the recommended values. These results indicated that honey samples from subtropical countries contain acceptable HMF levels unless they were kept for one year or more, at which time HMF concentrations will increase above recommended limits. This is of particular interest because HMF is reported to have genotoxic effects and mutagenic potential (Janzowski et al., 2000) and is widely consumed by humans. Previous studies on the formation of HMF in honey samples have also reported a considerable increase in its concentration when honey is stored at room temperature or as a result of heat treatment (Hase et al., 1973; Consentino et al., 1996; Langridge, 1977; Singh and Bath, 1998). At its best, honey should be consumed within six months following its harvesting (Turhan, 2009). Kalbov et al. (2003) showed that the HMF content in honey increases gradually during its storage, eventually exceeding the recommended values, as we observed. In addition to these facts, the HMF level in honey is dependent on the type of sugar present in honey, as well as the fructose: glucose ratio (Doner, 1977). The HMF formation results from the acid-catalyzed dehydration of hexose sugars with fructose being particularly susceptible. Fructose is reportedly unstable at pH 4.6 and is ve times more reactive than glucose (Lee and Nagy, 1990). Table 3 also showed that Tualang honey 1 recorded a signicantly higher HMF reading (1131.76 mg/kg) when compared to Tualang honey 2 (986.57 mg/kg), which was stored for a similar period (p < 0.05). However, the difference between the two was that Tualang honey 1 was non-radiated while Tualang honey 2 was radiated about 3 months prior to the analysis. It is possible that radiation reduced HMF formation by reducing the number of microorganisms that may accelerate its formation and reduce the honeys freshness. Therefore, radiation is an important step in maintaining the purity of honey. However, the content of HMF in nonevaporated samples (Tualang honey 3 and Gelam honey 1) was similar to that of evaporated samples (Tualang honey 4 and Gelam honey 2) when the storage period was similar (up to six months). This indicated that evaporation did not have a signicant effect on HMF formation when similarly stored honey samples were compared. The regression analysis (Table 4) clearly indicated that HMF content in honey samples signicantly correlated with storage duration, pH, free acids, lactones and total acidity. Storage duration showed a very strong correlation (R2 = 0.912; F value = 93.16) with HMF level, indicating that time is the most important factor that affects HMF formation. The other physicochemical parameters such as free acids and total acidity showed strong correlations with

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HMF formation, while pH and lactones showed only moderate correlations. Since both experimental data and statistical analysis indicated that storage duration, free acids, total acidity, honey pH and lactones levels signicantly correlated with HMF formation, conducting additional tests such as measuring free acids and total acidity may provide ways to quickly assess honey quality. 5. Conclusions We concluded that HMF concentrations in fresh Malaysian honey samples (stored within six months) (2.8024.87 mg/kg) were within the internationally recommended range (80 mg/kg for tropical honey). However, the same honey samples when stored from 12 to 24 months have much higher HMF concentrations that exceed the recommended levels that are considered to be suitable and safe for human consumption (118.471139.95 mg/kg). HPLC is the preferred method for HMF determination. Storage duration correlated very strongly with HMF formation. Free acids and total acidity strongly correlated with the HMF content of honey, whereas pH and lactones showed only moderate correlation with HMF content. Therefore, these physicochemical parameters can serve as additional parameters that facilitate quick assessments of honey purity and complement HMF readings as quality indicators of the honeys freshness. It is recommended that honey samples be consumed within six months to one year, regardless of the honey type. Acknowledgements This study was nancially supported by a grant from the Universiti Sains Malaysia RU (Grant No. 1001/PPSP/8120201). The authors would like to acknowledge the Federal Agricultural Marketing Authority (FAMA), Ministry of Agriculture and Agro based Industry, Malaysia for supplying Tualang honey and Rural Development Corporation, Sabah for supplying Borneo Tropical honey samples for this study. References
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