Determination of Adulterants in Honey Using HPLC Method

Determination of Adulterants in dearest Using HPLC MethodDetermination of adulterants such as hydroxymethylfurfural (HMF) in dearest victimisation HPLC method2. Materials and Methods2.1 MaterialsStandard of hydroxymethylfurfural was purchased from sigma Aldrich. All the samples and mensurations were diluted using distilled deionised urine.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 employ are stainless steel dish, porcelain crucible, beakers (5, 25, one hundred and 250 mL), pipettes (1, 10 and 50 mL), burette (10 and 25 mL), measuring cylinders (10, one C, 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 utilize were of make Gallenkamp, Memmert and respectivelyThe absorbance was recorded on a Perlong DNM-9602 Microplate Reader.2.2 Description of honey sample13 beloved samples of polar brands (Wescobee, Elodie, EL BREZAL, Hosen, Tropic, Lune de miel, Mc Mahons, ALSHIFA, Casino, Sunny, Gittos, Rodrigues Honey, and Natural Honey) were bought from Intermart Hypermarket, Jumbo Hypermarket and Monoprix Hypermarket.Brief details of different honey psychoanalysed, highlighting the manufacturing and expiry accompaniment, lot no, country of origin, and ingredients (if specified) are prone in table 5.Table 5 Description of honey samples experiment codeSample Brand/ NameDetailsSP1(a) Wescobee 100% Pure HoneyManufacturing age May 2014 breathing out date May 2017Lot no4107A agate line Australia fragment Pure honeySP9(b) Elodie Miel FruitManufacturing date NAExpiry date 28.08.15Lot no 4059IDOrigin France part NASP3(c) EL BREZAL Eucalyptus HoneyManufacturing date February 2014Expiry date February 2017Lot no 2247Origin SpainIngredien t 100% pure natural honeySP8(d) Hosen Pure HoneyManufacturing date 25.10.13Expiry date 24.10.15Lot no NAOrigin chinawareIngredient 100% honeySP6(e) Tropic Lychee HoneyManufacturing date September 2012Expiry date August 2014Lot no TP-002Origin IndiaIngredient Pure honeySP10(f) Lune de miel Miel de fleurManufacturing date NAExpiry date 01.05.16Lot noL28663AOrigin FranceIngredient 100% pure honeySP5(g) Mc Mahons Pure HoneyManufacturing date NAExpiry date March 2016Lot no B140813Origin AustraliaIngredient NASP7(h) ALSHIFA Natural HoneyManufacturing date November 2013Expiry date October 2018Lot no NAOrigin Saudi ArabiaIngredient 100% naturalSP4(i) Casino Miel de fleurManufacturing date NAExpiry date 10.03.16Lot no 206354Origin FranceIngredient NASP12(j) Sunny Pure HoneyManufacturing date NAExpiry date 24.01.16Lot no NAOrigin MauritiusIngredient HoneySP11(k) Gittos Special HoneyManufacturing date 27.06.14Expiry date 26.06.15Lot no NAOrigin MauritiusIngredient work over sugar syrup 85%, pure honey 15%SP2(l) Rodrigues HoneyManufacturing date NAExpiry date NALot no NAOrigin RodriguesIngredient NASP13(m) Natural HoneyManufacturing date NAExpiry date NALot no NAOrigin MauritiusIngredient NA2.3 Methods2.3.1 Sample preparation prior to physicochemical analysis2.3.1.1 MoistureMoisture was determined check to AOAC method (925.45D) (Appendix I). Stainless steel dish with 25g spinal column and a glass rod were dried in an oven for 1 hour, sereneed in a desiccator then weighed. 1g of homogenised honey sample was added and mixed thoroughly with the lynchpin by means of the rod. The latter was then enkindleed on steam bath for 15 min and dried in an oven for 2 hours at 60C, removed, allowed to cool in desiccator and weighed to a constant mass.2.3.1.2 AshAsh 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 wry on a water bath and heated on a hot plate until carbonized. The resulting carbonized samples were place in furnace at 600C for 6 hours, removed, allowed to cool in desiccator and weighed.2.3.1.3 pHpH was measured at 25C 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 AcidityFree 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 follows10g of honey samples were weighed in a 250 mL beaker and dissolved in 75 mL of CO2 free distilled deionised water (obtained by modify 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 mensurable as follows2.3.1.6 Electrical conductivityThe electrical conductivity was determined based on a method derived from Apiservices from the ash content of the honey samples according to the equation2.3.2 Sample preparation for HMF determination prior to HPLC analysisAliquots of honey samples were prepared by weighing 1g of honey and were diluted to 10 ml with distilled water, sieveed on 0.45 mm filter and injected into an HPLC equipped with a UV detector. The HPLC column was an Agilent, C18, 5m, 125 x 4 mm. The HPLC conditions were the following isocratic mobile phase, 1% of acetic acid and methanol in the ratio (8020) flow rate, 0.25 ml/min injection volume, 2 l, temperature 30C. 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 exemplification 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 sta ndard with that of one honey samples. The heart 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 (2530C) in a tumesce 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 40C, 60 C, 80C, and 100C for 5 min. The time was calculated when temperature reached the required degree. The honey samples were then cooled rapidly to room temperature (25C) and proceed as above to determine the HMF content.2.3.2.1 Calibration curve for HMF for HPLC analysisA 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 HMFConcentration/ppmVolume of 100 ppm stock used/mL101.00202.00303.00404.00505.00A 10 ppm spike sample of HMF was prepared by pipetting 200 L 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 quantificationThe 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 31. Different standards of HMF was analysed videlicet 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 101 was calculated by multiplying the obtained standard deviation by 10.2.3.3 Anti-oxidative property using DPPH radical scavenging activity4.5mg of DPPH (1, 1-diphenyl-2-picrylhydrazyl) was dissolved in 100 mL methanol and wrapped in aluminium peril to prevent light from entering. For the assay, a 96 wells Elisa plate was used. 100L of test sample was placed in the first well using micropipette. 50L methanol was added to all other wells and serial dilution was done. 50L of sample from the first well was pipette and transferred to the second well previously containing 50L methanol and the solution was mixed to ensure homogeneity. The 50L of the resulting solution was pipette and transferred to the third well and so on. Each well now contained 100L of solution after the dilution. 100L DPPH (4.5mg/100ml) solution was then added to every well. The solutions were incubated for 30 minutes at 37C 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 followsAntioxidative property of the samples firstly with no modify of the honeys and secondly with a heating temperature of 100C 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.1311