Determination and Comparision of Hydroxymethylfurfural in Industrial and Traditional Date Syrup Products

Background: Hydroxymethylfurfural (HMF) is a common Maillard reaction product directly formed from dehydration of sugars under acidic conditions during heating and storage in carbohydrate rich foods. The aim of the present study was to detect and quantify the amount of HMF in date syrup by HPLC method. In addition, the amount of HMF in date syrup produced by traditional and industrial methods were compared. Methods: A HPLC method for determination of HMF in date syrup was developed and validated. The amount of HMF in date syrup products produced by the traditional and industrial methods was compared. In addition, to determine whether HMF was produced during storage in date syrup, its amount was measured in fresh and old samples. Results: The HMF content of fresh traditional products varied between 1000 to 2675 mg⁄kg and in the old products varied between 2580 to 6450 mg⁄kg. The HMF concentration of the fresh industrial products varied between 12 to 456 mg⁄kg and 611 to 943 mg⁄kg in the old ones. The HMF concentration of the traditionally produced products was significantly higher than industrial products (P<0.001). Moreover, the HMF content in old products significantly was more than fresh ones (P<0.001). Wide variation was found in HMF content of the traditionally produced products. Conclusion: HMF was produced in date syrup during preparation and storage. Because HMF is recognized as an indicator of quality deterioration in a wide range of foods and is still under investigation for possible toxic effects, it is recommended that the amount of HMF is measured in date syrup and considered as an indicator of the quality control of this product.


INTRODUCTION
Date syrup is one of the most common date fruit products.It is a tick-dark liquid extracted by water from date fruit [1].The main component of date syrup is carbohydrate (about 70%).Glucose and fructose are the major sugar in the date syrup, besides, it contains some micronutrients such as iron, sodium, potassium, calcium, vitamin etc [2][3][4].It is a rich nourishment and suitable source for energy.The date syrup is commonly produced in date producing countries such as Iran.It is both directly consumed as a food and used for producing beverages, biscuits, ice cream, baby food and cakes as a sweetening and flavouring agent [1].Date syrup is mostly produced in traditional methods in Iran.However, as consumption of this product is increasing, it also has been produced by industrial units.In the traditional method for producing date syrup, date fruit and water are mixed and heated to boiling, filtered and then heated again to boiling until the desire concentration is obtained, but in industrial method the filtrate is concentrated under controlled heating and the temperature is not exceeded above 70 C.
During heating of carbohydrate-rich foods at high temperature, sucrose is decomposed into furfural compounds by two possible pathways: the caramelization and the Maillard reaction.5-Hydroxymethyl-2-furfuraldehyde (HMF) is a common product of these reactions [5,6].
HMF has been identified in a wide variety of heat-processed foods.Depending of production technology and storage, HMF content in foods varies considerably.While HMF is practically not present in fresh and untreated foods, high levels can be found in dried fruits, coffee and caramel products and in some foods the level of HMF exceed 1g/kg [7,8].Spontaneous production of HMF during the storage has been reported for some foods such as honey and jam [9,10].HMF is a good indicator for quality deterioration following overheating and storage of carbohydrate containing foods.For example, the Codex Alimentarius standard sets a maximum limit of 40 mg/kg for HMF in honey as a way of assuring that the product has not undergone heating during processing [11].
The presence of HMF in food has raised concern in relation to human health.This compound and its metabolite (5sulfidemethylfurfural) have genotoxic, mutagenic and carcinogenic effects in cell based and animal studies [7,[11][12][13][14].The date syrup is a carbohydrate-rich food product and is a suitable food matrix for production of HMF during production and storage.
The aim of the present study was to detect and quantify the amount of HMF in date syrup by HPLC method.In addition, the amount of HMF in date syrup produced by traditional and industrial methods were compared.

Samples
The samples were collected from different local markets in Jahrom City, Fars Province, Iran.These samples consisted of traditionally produced date syrup (n=40) and industrially produced date syrup (n=12).The industrial products were from two food production companies which produce date syrup in Iran.Besides, samples included old products with production date of more than 6 month and fresh products with production date of less than 1 month.

Sample Preparation
0.5 gram of homogenised date syrup was dissolved in 500 ml ultra-pure water and filtered through a 0.2 µm membrane filter before injection into the HPLC column.

HPLC System and Condition
The HPLC system consisted of binary pump, degasser, sampling valve, 20 µl sample loop, column oven, photo-diode array detector and computing integrator connected to a PC, all from Shimadzu (Japan).The mobile phase was a mixture of acetonitrile in water (15% v/v) delivered at a flow rate of 1 ml/min under isocratic condition through the analytical column, Eurospher-100 C18 (5 µm, 4.6 × 250 mm).The chromatogram was monitored at 285 and the volume of injection was 20 µl.The column oven temperature was set on 30 C.HMF was quantified using the external standard method.

Statistical Analysis
Statistical significance of data was tested by non-parametric Mann Whitney test between groups.Analyses were performed by Graph Pad Prism software version 5.  The external calibration curve was used for quantification of HMF.For assessing of matrix effect, the calibration curve of spiked samples was compared with calibration curve of aqueous standard solution in concentration range of 0.2-2 µg/ml.The slope of the regression line for the calibration curve of spiked samples and aqueous standard solution was the same.Then the calibration curve of aqueous standard solution was used for the quantification of HMF in the samples.The validation parameters are represented in Table 1.Linearity was determined in concentration range of 0.2-2 µg/ml and correlation coefficient (R 2 ) of 0.99 was found indicating a good linear relationship between concentration and peak area.The limit of detection (LOD) and limit of quantification (LOQ) were determined by following formula: LOD= 3.3SD/m, LOQ= 10SD/m where SD is residual standard deviation of regression line and m is the slope of regression line.Intra-day precision was determined by replicate analysis (n = 6) of the spiked sample on the same day and inter-day precision was determined by replicate analysis of the spiked sample on six consecutive days.Assay precision was expressed as the relative standard deviation (RSD %).Intra-day and inter-day precisions for different concentration were between 1-3% and 1-6%, respectively.The recoveries for spiked samples at different concentrations (0.2-2µg/ml) were between 97-102%.These data are in acceptable ranges described in the AOAC manual for peer-verified methods.

Comparison of HMF Content in the Date Products
Comparison of average HMF contents of industrial and traditional products is shown in Fig. 2. Amount of HMF in traditional products significantly was more than industrial products (P<0.05).The amount of HMF in traditional products varies between concentrations of 1000 to 2675 mg⁄kg in the fresh and 2580 to 6450 mg⁄kg in the old products.The amount of HMF in the industrial products varies between concentration of 12 to 456 mg⁄kg in fresh, and 611 to 943 mg⁄kg in the old ones.The wide variation was observed between traditional products (Fig. 3).Fig. 4 shows comparison of average HMF content between old and fresh products.The amount of HMF was significantly higher in old products rather than fresh ones (P<0.05).

Table 1.
Validation parameters data for the HMF quantified in date syrup.

DISCUSSION
In the present study the amount of HMF in date syrup was quantified by HPLC method.HMF was determined and quantified in various food stuffs such as honey, bakery products, jams, dried fruits and fruit juices by HPLC method.
In the present study the amount of HMF in the 40 traditionally and 12 industrially produced date syrup was measured.The wide variation was observed between HMF content of traditional products.Moreover, the amount of HMF in traditional products was significantly greater than industrial products.These differences may be due to lack of control on temperature, boiling, and long duration of heating in traditional methods.In traditional method the date juice is boiled for a long time to meet desire concentration and a long http://www.ijt.ir;Vol 10, No 5, September-October 2016 time of evaporation process is necessary, but in manufacturing processes the date juice is evaporated under vacuum in controlled low temperature condition then the juice is concentrated during less time.Overheating of carbohydrate-containing foods produces more HMF.Higher amount of HMF was produced in palm sugar syrup when high temperature (110 ̊ C) processing method was used [15].The same result was reported for other foods such as bakery product [16] and the formation of HMF is dependent on time of process in boiling juice.Highly variable amount of HMF, changing from 12.8 to 3500 µg/kg, were reported for the boiled juices.Among them HMF concentration of boiled pomegranate juice was the highest (514-3500 µg/kg).
Our result indicated that the amount of HMF was significantly higher in old product rather than fresh ones.This result implies that HMF is continuously produced during the storage of date syrup.The similar findings were reported for other food commodities.The amount of HMF significantly increases in honey [10], jams and fruit-based infant foods [9] one year after production.Dehydration of carbohydrate produces HMF during storage.The temperature of storage condition is an important factor for production of HMF in food product.Storage of food products at high temperatures causes to produce more HMF.

CONCLUSION
There is no survey about HMF content of date syrup and our study is the first investigation.Date syrup is a suitable matrix for production of HMF and traditional method is not a proper method for preparation of this food product.HMF can be a good indicator of quality deterioration during production and storage of date syrup.Because the date syrup is highly consumed in date-producing areas like south of Iran, high levels of HMF in traditional products can threaten the health of consumers of this product.Then it is recommended that amount of HMF in date syrup is regulated.

Fig. 1
Fig. 1 represents the chromatogram of date syrup sample spiked by 1 µg/ml HMF in optimized HPLC condition.The optimized condition achieved by acetonitril/water ratio of 15/85 and flow rate 1 ml/min.In this condition the sharp and pure peak for HMF was appeared in reasonable retention time (4.7 min).

Figure 1 .
Figure 1.The chromatogram of date syrup sample which spiked by 1 µg/ml HMF.

Figure 4 .
Figure 4. Comparison of HMF content of old and Fresh products, industrially produced date syrup products (A)and traditionally produced date syrup products (B) ***p<0.001.