1、ICS 67.180.10Untersuchung von Honig Bestimmung des Gehaltes an Hydroxymethylfurfural Teil 3: Hochleistungs-flssigchromatographisches VerfahrenIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the de
2、cimal marker.Ref. No. DIN 10751-3 : 2002-02English price group 08 Sales No. 010811.03DEUTSCHE NORM February 200210751-3 No part of this translation may be reproduced without the prior permission ofDIN Deutsches Institut fr Normung e.V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclus
3、ive right of sale for German Standards (DIN-Normen).Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.Determining the hydroxymethylfurfural contentof honey by high-performance liquidchromatographyContinued on pages 2 to 5.F
4、orewordThis standard has been prepared by Technical Committee Honiguntersuchung of the NormenausschussLebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Commit-tee).1 ScopeThis standard specifies a method of determining the content of hydroxymethylfurfural
5、(HMF) and othersubstances in honey.NOTE: The method can be used as an alternative to determining the hydroxymethylfurfural content by theWinkler photometric method or by the White direct photometric method specified in DIN 10751-1 andDIN 10751-2.2 ConceptHydroxymethylfurfural contentThe content of h
6、ydroxymethylfurfural (5-hydroxymethyl-2-furaldehyde) in honey, in mg/kg, determined byhigh-performance liquid chromatography (HPLC) as described in this standard.3 PrincipleAfter simultaneous clarification and stabilization with Carrez solution, a test solution is filtered and, afterseparation by hi
7、gh-performance liquid chromatography, the hydroxymethylfurfural content is determinedusing a UV detector at a wavelength of 284 nm. Quantitative results are obtained by the external standardmethod from the peak heights or peak areas of the test and calibration solutions, while their retention timesa
8、re used for identification purposes.Page 2DIN 10751-3 : 2002-024 ReagentsOnly analytical grade reagents shall be used and the water used shall be distilled water or water of at leastequivalent purity. The term solution means an aqueous solution.The following reagents shall be used.4.1 Double-distill
9、ed water, suitable for HPLC.4.2 0,01 mol/l disodium hydrogenphosphate solution, Na2HPO4.4.3 Methanol, suitable for HPLC analysis.4.4 85% (V/V) and 10% (V/V) orthophosphoric acid solutions, H3PO4.4.5 High-purity potassium hexacyanoferrate(II) trihydrate, K4Fe(CN)6 3H2O.4.6 Zinc acetate dihydrate, Zn(
10、CH3COO)2 2H2O.4.7 5-Hydroxymethyl-2-furaldehyde, C6H6O3.4.8 HMF standard solution, prepared by weighing 10 mg of HMF into a 100 ml volumetric flask, dissolvingin water, making up to the mark and shaking.To determine the HMF content of this solution exactly, pipette 5 ml of it into a 100 ml volumetri
11、c flask, makeup to the mark with water and determine the content of this solution photometrically at a wavelength of 284 nm,with water as reference. Calculate the HMF content, rHMF, in mg/l, using equation (1):E284 M 1000 F E284 126 1000 20 E284 252rHMF= = = (1)e d 16830 1 1,683whereE284is the absor
12、bance at 284 nm;e is the molar absorptivity at 284 nm (here, e = 16830);d is the path length of the cell (here, d = 1 cm);M is the molar mass of HMF (here, M = 126);F is a dilution factor (here, F = 20).If frozen, the standard solution will be stable for several years.4.9 HMF calibration solutions,
13、containing between 0,2 g/ml and 20 g/ml HMF, prepared by diluting thestandard solution. Prepare four or five solutions.4.10 Mobile phase, prepared by adjusting the pH value of the disodium hydrogenphosphate solution to 3,using the 85% (V/V) and 10% (V/V) phosphoric acid solutions, respectively, for
14、the coarse and fine adjust-ments, and then mixing 95 parts by volume of this solution with five parts by volume of methanol.4.11 CarrezI-solution, prepared by dissolving 15 g of potassium hexacyanoferrate(II) trihydrate in waterin a 100 ml volumetric flask and making up to the mark with water.4.12 C
15、arrez-II- solution, prepared by dissolving 30 g of zinc acetate dihydrate in water in a 100 ml volumet-ric flask and making up to the mark with water.5 ApparatusIn addition to standard laboratory equipment, the following shall be used.5.1 Screen, made of stainless steel, with an aperture size of 0,5
16、 mm.5.2 Beakers, of nominal capacities 50 ml and 100 ml.5.3 Volumetric flasks, of nominal capacities 50 ml and 100 ml.5.4 Filter paper, analytical grade.5.5 pH meter.5.6 UV spectrometer, suitable for measurements at 284 nm.5.7 Quartz cells, with a path length of 1 cm.5.8 Test tubes, of nominal capac
17、ity about 30 ml.Page 3DIN 10751-3 : 2002-025.9 Membrane filter, of pore size 0,45 m.5.10 HPLC chromatograph, having a pump or pumps, an injection system suitable for a sample volume of1l to 100 l, a UV detector and an evaluation system.5.11 Separation column1), having an internal diameter of 3 mm to
18、 5 mm and a length of 250 mm, packed withLichrospher 100 RP 182) having a particle size of 5 m and, if necessary, fitted with a precolumn.6 SamplingPending publication of an appropriate sampling standard, the procedure shall be subject to agreement. How-ever, a representative laboratory sample of no
19、t less than 200 g shall be used for the analysis.7 Procedure7.1 Sample preparation7.1.1 Pure liquid or set honeyHomogenize the laboratory sample sufficiently by stirring vigorously for not less than three minutes, taking careto minimize the amount of air included.7.1.2 Impure liquid or set honeyAfte
20、r removing coarse impurities, stir the honey at ambient temperature until smooth and pass through a screen,using a spatula in the case of set honey.7.1.3 Comb honeyIf the combs are still sealed, first remove the cover, then separate the unheated honey completely from thecombs using a screen.7.2 Prep
21、aring the test solutionWeigh, to an accuracy of 0,1 g, 10 g of honey into a beaker, dissolve it in 50 ml of distilled water and transferthe solution quantitatively to a 100 ml volumetric flask. To precipitate the proteins and stabilize the HMF, add1ml each of Carrez-I-solution and Carrez-II-solution
22、, make the solution up to the mark with water, mixthoroughly and filter through a filter paper. Then filter through a membrane filter, if considered necessary, butbear in mind that HMF losses may result from the use of such filters. Finally, apply the filtrate to the injectionsystem.NOTE: After trea
23、tment with the Carrez solutions, the test solution will be stable for up to 14 hours.7.3 High performance liquid chromatographyThe following procedure and settings have proved satisfactory for the column specified in subclause 5.11 if theelution solution specified in subclause 4.10 is used.Flow rate
24、: 1 ml/minMobile phase: As in subclause 4.10Volume injected: 10 l to 50 lWavelength for HMF detection: 284 nm.7.4 AnalysisTo obtain a quantitative result, use the external standard method and integrate the peak areas or determine thepeak heights from at least two injections.To prepare a calibration
25、graph, inject the HMF calibration solutions, plot the quantity injected, in g/ml, as afunction of the peak areas or heights found and determine the best-fit straight line graphically or by calculation.NOTE: The volumes of the test and HMF calibration solutions should be the same.Peaks are deemed to
26、be incompletely separated (2% overlap) if R in equation (2) is equal to unity, but to becompletely separated if R equals 1,25.1) Information on suppliers of suitable columns is obtainable from the Normenauschuss Lebensmittel undlandwirtschaftliche Produkte of DIN, 10772 Berlin, Germany.2) Lichrosphe
27、r 100 RP 18 is an example of a suitable commercial product. It is mentioned solely as guidancefor the user of this standard without this implying any recognition of the product by DIN.Page 4DIN 10751-3 : 2002-02tR2 tR1tR2 tR1R = 2 = 1,8 (2)W1+ W2 W1/2 1+ W1/2 2wheretRis the retention time, i.e. tR2
28、tR1 is the difference in the retention times for two peaks;W is the peak width, i.e. W1 + W2is the sum of the peak widths;W1/2is the peak width at mid-height, i.e. W1/2 1+ W1/2 2is the sum of the peak widths at mid-height.8 Evaluation8.1 CalculationIf equal volumes of the test and HMF calibration so
29、lutions were injected, the HMF content of honey, vHMF, inmg/kg, is given by equation (3):mst 100vHMF= (3)mPrwheremstis the mass obtained from the best-fit straight line for the calibration solution, in g;mPris the initial sample mass, in g.Report the result to three significant places.8.2 Precision8
30、.2.1 Interlaboratory testingAn interlaboratory test carried out on four honey samples with different HMF contents yielded the results shownin Appendix A.8.2.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability conditi
31、ons will not exceedthe repeatability limit, r, in more than 5% of cases.Values obtained from peak area evaluation:Sample no. 1: x_= 4,12 mg/kg r = 0,42 mg/kgSample no. 2: x_= 14,71 mg/kg r = 0,99 mg/kgSample no. 3: x_= 20,75 mg/kg r = 1,40 mg/kgSample no. 4: x_= 38,81 mg/kg r = 1,55 mg/kgValues obta
32、ined from peak height evaluation:Sample no. 1: x_= 3,97 mg/kg r = 0,36 mg/kgSample no. 2: x_= 14,46 mg/kg r = 1,11 mg/kgSample no. 3: x_= 20,43 mg/kg r = 1,45 mg/kgSample no. 4: x_= 38,15 mg/kg r = 1,50 mg/kg8.2.3 Reproducibility limit(different operators, different equipment)The absolute difference
33、 between two individual results obtained under reproducibility conditions will notexceed the reproducibility limit, R, in more than 5% of cases.Values obtained from peak area evaluation:Sample no. 1: x_= 4,12 mg/kg R = 1,84 mg/kgSample no. 2: x_= 14,71 mg/kg R = 4,26 mg/kgSample no. 3: x_= 20,75 mg/
34、kg R = 5,08 mg/kgSample no. 4: x_= 38,81 mg/kg R = 7,74 mg/kgValues obtained from peak height evaluation:Sample no. 1: x_= 3,97 mg/kg R = 1,47 mg/kgSample no. 2: x_= 14,46 mg/kg R = 3,86 mg/kgSample no. 3: x_= 20,43 mg/kg R = 4,64 mg/kgSample no. 4: x_= 38,15 mg/kg R = 7,51 mg/kgPage 5DIN 10751-3 :
35、2002-029 Test reportThe test report shall refer to this standard and include the following details as a minimum:a) type, origin and designation of sample;b) type and date of sampling;c) dates of receipt and testing;d) HMF content of sample, in mg/kg;e) reason for any deviation from this standard.App
36、endix AResults of interlaboratory testingAn interlaboratory test carried out by Technical Committee Honiguntersuchung and evaluated as specified inISO 5725-1 and ISO 5725-2 yielded the results listed in tables A.1 and A.2 (all values are given in mg/kg).Table A.1: Precision data from peak area evalu
37、ationBibliographyISO 5725-1 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 1: Generalprinciples and definitionsISO 5725-2 : 1994 Accuracy (trueness and precision) of measurement methods and results Part 2: Basicmethod for the determination of repeatability and repro
38、ducibility of a standard method ofmeasurementMeyer, V., Praxis der Hochleistungschromatographie (High-performance chromatography practice), 7thed.,Frankfurt/Main: Otto-Salle-Verlag; Frankfurt/Main, Salzburg: Verlag Sauerlnder Aarau, 1992, 22.Parameter Sample 1 Sample 2 Sample 3 Sample 4Mean, x_4,12
39、14,71 20,75 38,81Repeatability limit, r 0,42 0,99 1,40 1,55Repeatability standard deviation, sr0,15 0,35 0,50 0,55Reproducibility limit, R 1,84 4,26 5,08 7,74Reproducibility standard deviation, sR0,65 1,51 1,79 2,74Table A.2: Precision data from peak height evaluationParameter Sample 1 Sample 2 Sample 3 Sample 4Mean, x_3,97 14,46 20,43 38,15Repeatability limit, r 0,36 1,11 1,45 1,50Repeatability standard deviation, sr0,13 0,39 0,51 0,53Reproducibility limit, R 1,47 3,86 4,64 7,51Reproducibility standard deviation, sR0,52 1,36 1,64 2,66
