ABSTRACT
HMF (5-hydroxymethylfurfuraldehyde) is essential to evaluate the conformity of honey to the current legislation. Elevated concentration of HMF in honey provides an indication of overheating, storage in poor condition or age of the honey. As per Food Safety Standard Authority of India (FSSAI), the HMF concentration in honey should not exceed 80 mg/kg. The aim of this study is to estimate and compare HMF content in different marketed brands of honey samples by using two spectrophotomteric methods, namely, White’s method and Winkler’s method. Both the methods showed comparable results in all the honey samples, however, Winkler’s method showed a little higher value than White’s method. Both the methods showed excellent recovery of HMF. White’s method was found to be cost effective, less time consuming and less hazardous than Winkler’s method.
KEYWORDS: HMF (5-hydroxymethylfurfuraldehyde), Honey, Spectrophotometry.
INTRODUCTION
Honey is a sweet natural product produced by honeybees (Apis mellifera), which gather nectar from flowers before converting it to nutritious food. Honey is considered both nutritional and medicinal, although the presence of certain constituents, for example, heavy metals (even in trace amounts), some alkaloids, and HMF and its derivatives may contribute to honey’s toxicity. HMF is a cyclic aldehyde produced by sugar degradation through the Maillard reaction (a nonenzymatic browning reaction) during food processing or long storage of honey. The presence of simple sugars (glucose and fructose) and many acids, as well as minerals, in honey can further enhance the production of this substance. HMF concentration is widely recognized as a parameter affecting honey freshness because it is typically absent (or is present in only very small amounts in fresh honeys), while its concentration tends to rise during processing and/or because of aging.
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 European 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).
In India, the primary regulatory authority, the Food Safety Standard Authority of India (FSSAI) established that the HMF content of honey must not be higher than 80 mg/kg.
The International Honey Commission (IHC, Stefan Bogdanov, 1999, pp. 1–54) recommends two spectrophotometric methods for the determination of HMF. These two spectrophotometric methods, determination after White (1979) and after Winkler (1955). 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 present study is designed to estimate the HMF content in different marketed honey samples using both these method and to compare the variations of results among them. Additionally recovery study of HMF was also been carried out using both these two methods.
MATERIALS AND METHODS
Samples, Chemicals and reagents: Honey samples of different brands were obtained from local market of Kolkata. All the chemicals and reagents are of AR grade, HMF standard, Sodium bi sulphite, barbituric acid and p-toluidine were obtained from Merck, India.
White’s method for determination of HMF: 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 Shimadzu UV-1900 spectrophotometer. The quantitative value of HMF was determined both by the external standard method and by using the proposed formula for the method reported by IHC (IHC, Stefan Bogdanov, 1999, pp. 1–54).
Calculation and Expression of results:
HMF in mg/kg = (A284 – A336) x 149.7 x 5 x D/W
Where:-
A284 = absorbance at 284 nm
A336 = absorbance at 336 nm
126 x 1000 x 1000
149.7 = —————————– = Constant
16830 x 10 x 5
126 = molecular weight of HMF
16830 = molar absorptivity ε of HMF at λ= 284 nm
1000 = conversion g into mg
10 = conversion 5 into 50 ml
1000 = conversion g of honey into kg
5 = theoretical nominal sample weight
D = dilution factor, in case dilution is necessary
W = Weight in g of the honey sample
Express results in mg/kg to 1 decimal place.
Winkler’s method for determination of HMF 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 Shimadzu UV-1900 spectrophotometer. The quantitative value of HMF was determined both by the external standard method and by using the proposed formula for the method (IHC, Stefan Bogdanov, 1999, pp. 1–54).
Calculation and Expression of results:
192xAx10
HMF in mg/kg = ————————————
Weight of honey in grams
Where,
A = Absorbance,
192 = Factor for dilution and extinction coefficient
Express results in mg/kg to 1 decimal place
RESULTS AND DISCUSSION
Estimation of HMF Content in Honey
HMF content of different brands of honey samples was estimated by both the spectrophotomteric method. As shown in Table 1 and Figure 1, HMF contents were varied. This phenomenon might be attributed to the varied processing techniques and ageing in these honey samples. The analyses were carried out in triplicates.
Recovery Study
Three different doses of HMF was added to the honey samples and estimated by both Winkler’s and White’s method. The recovery percentage was depicted in table 2, 3 and Figure 2.
Table 1: Result of HMF content by Winkler’s method and White’s method
| Samples | Results in mg/kg (Mean+SD) | |
| By Winkler’s Method | By White’s Method | |
| Brand 1 | 23.64+1.41 | 21.92+1.10 |
| Brand 2 | 20.65+0.66 | 18.89+0.30 |
| Brand 3 | 14.36+0.96 | 12.89+0.78 |
| Brand 4 | 55.41+1.37 | 53.75+1.27 |
| Brand 5 | 78.22+0.86 | 74.18+1.07 |
Figure 1: Result of HMF content by Winkler’s method and White’s method
Table 2: Recovery of HMF content by Winkler’s method
| Sample | % Recovery by Winkler’s method | |||||
| HMF Content (mg/kg) | Added (mg/kg) | Total (mg/kg) | Obtained (mg/kg) | % Recovery | Avg Recovery in % | |
| Brand 1 | 23.64 | 10 | 33.64 | 32.04 | 95.24 | 95.98+4.33 |
| 20 | 43.64 | 40.18 | 92.07 | |||
| 30 | 53.64 | 53.98 | 100.63 | |||
| Brand 2 | 20.65 | 10 | 30.65 | 28.79 | 93.93 | 93.86+0.66 |
| 20 | 40.65 | 38.41 | 94.49 | |||
| 30 | 50.65 | 47.19 | 93.17 | |||
| Brand 3 | 14.36 | 10 | 24.36 | 23.16 | 95.07 | 96.24+2.59 |
| 20 | 34.36 | 34.09 | 99.21 | |||
| 30 | 44.36 | 41.89 | 94.43 | |||
| Brand 4 | 55.41 | 10 | 65.41 | 63.74 | 97.45 | 96.16+1.83 |
| 20 | 75.41 | 73.19 | 97.06 | |||
| 30 | 85.41 | 82.81 | 96.96 | |||
| Brand 5 | 78.22 | 10 | 88.22 | 85.77 | 97.22 | 96.78+0.48 |
| 20 | 98.22 | 95.14 | 96.86 | |||
| 30 | 108.22 | 104.18 | 96.27 | |||
Table 3: Recovery of HMF content by White’s method
| Sample | % Recovery by White’s method | |||||
| HMF Content (mg/kg) | Added (mg/kg) | Total (mg/kg) | Obtained (mg/kg) | % Recovery | Avg Recovery in % | |
| Brand 1 | 21.92 | 10 | 31.92 | 31.24 | 97.87 | 97.99+0.37 |
| 20 | 41.92 | 40.95 | 97.69 | |||
| 30 | 51.92 | 51.09 | 98.40 | |||
| Brand 2 | 18.89 | 10 | 28.89 | 28.70 | 99.34 | 98.90+0.68 |
| 20 | 38.89 | 38.16 | 98.12 | |||
| 30 | 48.89 | 48.52 | 99.24 | |||
| Brand 3 | 12.89 | 10 | 22.89 | 21.85 | 95.46 | 97.26+1.67 |
| 20 | 32.89 | 32.09 | 97.57 | |||
| 30 | 42.89 | 42.36 | 98.76 | |||
| Brand 4 | 53.75 | 10 | 63.75 | 63.74 | 99.98 | 98.83+1.11 |
| 20 | 73.75 | 72.11 | 97.78 | |||
| 30 | 83.75 | 82.69 | 98.73 | |||
| Brand 5 | 74.18 | 10 | 84.18 | 83.71 | 99.44 | 98.95+0.47 |
| 20 | 94.18 | 93.14 | 98.90 | |||
| 30 | 104.18 | 102.63 | 98.51 | |||
Figure 2: Recovery of HMF content by White’s method
CONCLUSION:
This study dealt with two spectrophotometric methods, namely, Winkler’s method and White’s method for the estimation of HMF content of honey. Both the methods applied in the study allowed to achieve excellent recovery of the analyte. Average recovery was 95.80 % in the Winkler’s method and 98.39% in the White’s method. The values of percent recovery indicate that both tested analytical methods are suitable for determination of HMF content in honey. Owing to higher percent recovery of White’s method, cost of chemicals in Winkler approach, and carcinogenicity of p-toluidine applied in the determination process, White’s method seems to be a superior method for determination of HMF content in natural honey. The HMF content was determined in 5 different brands of honey samples. None of the honey samples showed HMF value greater than 80 mg/kg as stipulated by FSSAI. The above indicates that commercially available honey is characterized by satisfactory quality in terms of HMF content. The developed methods were simple, quick, accurate, and precise, do not require expensive instrumentation, and could be easily incorporated in routine analysis of Honey to meet the requirement of regulatory authorities.