Determination of Adulterants in Honey Using HPLC Method

2. Materials and Methods

2.1 Materials

Standard of hydroxymethylfurfural was purchased from sigma Aldrich. All the samples and standards were diluted using distilled deionised water.

Methanol, sodium hydroxide,diphenyl-1-pikryl, ascorbic acid, 2,2- hydrochloric acid and acetic acid were of analytical reagent grade and purchased from Techno PharmChem, Avonchem and Alpha Chemika Ltd.

Most glassware used are stainless steel dish, porcelain crucible, beakers (5, 25, 100 and 250 mL), pipettes (1, 10 and 50 mL), burette (10 and 25 mL), measuring cylinders (10, 100, 250 and 500 mL) and volumetric flasks (10, 100, 200, 1000 mL) were of grade A. All the glasswares were rinsed with distilled deionised water to remove surface contaminants prior to use.

Oven, Incubator and furnace used were of make Gallenkamp, Memmert and respectively

The absorbance was recorded on a Perlong DNM-9602 Microplate Reader.

2.2 Description of honey sample

13 Honey samples of different brands (Wescobee, Elodie, EL BREZAL, Hosen, Tropic, Lune de miel, Mc Mahon’s, ALSHIFA, Casino, Sunny, Gitto’s, Rodrigues Honey, and Natural Honey) were bought from Intermart Hypermarket, Jumbo Hypermarket and Monoprix Hypermarket.

Brief details of different honey analysed, highlighting the manufacturing and expiry date, lot no, country of origin, and ingredients (if specified) are given in table 5.

Table 5: Description of honey samples

2.3 Methods

2.3.1 Sample preparation prior to physicochemical analysis

2.3.1.1 Moisture

Moisture was determined according to AOAC method (925.45D) (Appendix I). Stainless steel dish with 25g sand and a glass rod were dried in an oven for 1 hour, cooled in a desiccator then weighed. 1g of homogenised honey sample was added and mixed thoroughly with the sand by means of the rod. The latter was then heated on steam bath for 15 min and dried in an oven for 2 hours at 60°C, removed, allowed to cool in desiccator and weighed to a constant mass.

2.3.1.2 Ash

Ash was determined according to AOAC method (920.181) (Appendix II) such that 5g of homogenized honey samples were added to pre-weighed empty porcelain. The samples were then allowed to dry on a water bath and heated on a hot plate until carbonized. The resulting carbonized samples were place in furnace at 600°C for 6 hours, removed, allowed to cool in desiccator and weighed.

2.3.1.3 pH

pH was measured at 25°C by preparing a 10% (w/v) solution (dry weight basis) in distilled deionised water by a Delta Ohm HD 8706 pH meter.

2.3.1.4 Total Acidity

Free acids, lactones, total acidity and pH were measured using a Mettler Toledo MP 220 pH meter according to the AOAC method 962.19 (Appendix III) as follows:

10g of honey samples were weighed in a 250 mL beaker and dissolved in 75 mL of CO₂ ^–free distilled deionised water (obtained by cooling freshly boiled deionised water). The mixtures were stirred using magnetic stirrer and titrated against 0.05M sodium hydroxide at a rate of 5 mL/min until the pH reached 8.50. 10 mL of 0.05M sodium hydroxide was pipetted and back-titrated with 0.05M hydrochloric acid to pH 8.30. A blank titration was also performed using similar procedure. Acidity of honey samples were calculated as follows:

2.3.1.6 Electrical conductivity

The electrical conductivity was determined based on a method derived from Apiservices from the ash content of the honey samples according to the equation:

2.3.2 Sample preparation for HMF determination prior to HPLC analysis

Aliquots of honey samples were prepared by weighing 1g of honey and were diluted to 10 ml with distilled water, filtered on 0.45 mm filter and injected into an HPLC equipped with a UV detector. The HPLC column was an Agilent, C18, 5µm, 125 x 4 mm. The HPLC conditions were the following: isocratic mobile phase, 1% of acetic acid and methanol in the ratio (80:20); flow rate, 0.25 ml/min; injection volume, 2 µl, temperature; 30°C. All the solvents were of HPLC grade. The chromatograms were monitored at 285 nm. All the samples were analysed in triplicates and after every 6 samples, a standard check was analysed. HMF was identified by analysing the peak in honey with a standard HMF, and by comparison of the spectra of the HMF standard with that of one honey samples. The amount of HMF in the honey samples was determined using a calibration curve and by comparing the peak area of the standard and the resulting samples.

All honey samples were stored at room temperature (25–30°C) in a well closed container and the HMF content of each sample was analysed on a monthly basis throughout a period of four months.

To determine HMF progress during heat treatment, honey samples were subjected to heat treatment by placing 1g honey sample in a glass container, and heat in a water bath at 40ºC, 60 ºC, 80ºC, and 100ºC for 5 min. The time was calculated when temperature reached the required degree. The honey samples were then cooled rapidly to room temperature (25°C) and proceed as above to determine the HMF content.

2.3.2.1 Calibration curve for HMF for HPLC analysis

A 100 ppm stock solution of HMF standard was prepared by dissolving 0.0101 g of HMF standard in 100 mL of distilled deionised water in a 100 mL volumetric flask. From the 100 ppm stock solution, 10 ppm, 20 ppm, 30 ppm, 40 ppm and 50 ppm standard solution were prepared separately in 10 mL volumetric flasks. The different volumes of the stock solutions which were diluted to 10 mL are given in Table 4. The resulting standards were analysed on a HPLC UV detector at 285 nm and a calibration curve was plotted.

Table 6: Volume of 100 ppm stock to prepare different concentration of HMF

A 10 ppm spike sample of HMF was prepared by pipetting 200 µL of 100 ppm stock solution of HMF standard and transferred to the 2 g sample of honey and diluted to 20 mL with distilled deionised water.

2.3.2.2 Limit of detection and quantification

The limit of detection and quantification of HMF was calculated according to EPA method SW-846 (Appendix V). LOD is defined to be the minimum level at which the analyte can be detected reliably with signal-to noise 3:1. Different standards of HMF was analysed namely 0.1, 0.05 and 0.04ppm such that detection limit of HMF was thus then established by analysing a 0.05ppm HMF standard solution seven times and the standard deviation of the repeats for the analyse was multiplied by a factor 3.14 based on student t-statistics. The limit of quantification with signal-to noise ratio 10:1 was calculated by multiplying the obtained standard deviation by 10.

2.3.3 Anti-oxidative property using DPPH radical scavenging activity

4.5mg of DPPH (1, 1-diphenyl-2-picrylhydrazyl) was dissolved in 100 mL methanol and wrapped in aluminium foil to prevent light from entering. For the assay, a 96 wells Elisa plate was used. 100µL of test sample was placed in the first well using micropipette. 50µL methanol was added to all other wells and serial dilution was done. 50µL of sample from the first well was pipette and transferred to the second well previously containing 50µL methanol and the solution was mixed to ensure homogeneity. The 50µL of the resulting solution was pipette and transferred to the third well and so on. Each well now contained 100µL of solution after the dilution. 100µL DPPH (4.5mg/100ml) solution was then added to every well. The solutions were incubated for 30 minutes at 37°C in an incubator and the absorbances of the resulting solutions were read at 492nm on a Perlong DNM-9602 Microplate Reader. The % scavenging activity of the samples was calculated as follows:

Antioxidative property of the samples; firstly with no heating of the honeys and secondly with a heating temperature of 100°C for five minutes were performed and proceed similarly to that of control ascorbic acid.

Note: A yellowish change in colour indicates the presence of ascorbic acid activity.

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