1、 DEUTSCHE NORM June 2004DIN 10763 ICS 67.180.10 Analysis of honey Determination of glycerol content Enzymatic method Untersuchung von Honig Bestimmung des Gehaltes an Glycerin Enzymatisches Verfahren Document comprises 11 pagesTranslation by DIN-Sprachendienst. In case of doubt, the German-language
2、original should be consulted as the authoritative text. No part of this translation may be reproduced without prior permission of DIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany, has the exclusive right of sale for German Standards (DIN-Normen). English pric
3、e group 9 www.din.de www.beuth.de !,inA“04.06 9707530DIN 10763:2004-06 2 Contents Page Foreword3 1 Scope 3 2 Normative references 3 3 Terms and definitions .3 4 Principle4 5 Reagents.4 6 Apparatus .5 7 Procedure .6 8 Calculation7 9 Reliability8 10 Precision.9 11 Test report 9 Annex A (informative) R
4、esults of interlaboratory testing 10 Bibliography. 11 DIN 10763:2004-06 3 Foreword This standard has been prepared by Technical Committee Honiguntersuchung of the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee). 1 Scope This st
5、andard specifies an enzymatic method of determining the glycerol content of honey. 2 Normative references This standard incorporates, by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text, and the titles of the p
6、ublications are listed below. For dated references, subsequent amendments to or revisions of any of these publications apply to this standard only when incorporated in it by amendment or revision. For undated references, the latest edition of the publication referred to applies (including any amendm
7、ents). DIN 12699:1975, Class AS fast delivery enzyme assay pipettes with a waiting time of 15 seconds, for laboratory use ISO 3696:1987, Water for analytical laboratory use Specification and test methods ISO 5725-1:1994, Accuracy (trueness and precision) of measurement methods and results Part 1: Ge
8、neral principles and definitions ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method 3 Terms and definitions For the purposes of this standard, the follo
9、wing terms and definitions apply. 3.1 glycerol content the glycerol content of honey determined by the method described in this standard and reported in mg/kg of sample material 3.2 specificity (cf. subclause 9.1) the ability to detect the analyte and distinguish it from similar substances, impuriti
10、es and degradation products 3.3 sensitivity the ability to detect small changes in the concentration of a substance in the test material 3.4 limit of detection the content to be regarded as the critical level for the quantity being measured if the calibration function determined is used DIN 10763:20
11、04-06 4 3.5 linearity the linear range is that concentration range of the analyte in which the results found by the method are proportional to its concentration 4 Principle Glycerol is phosphorylated to form L-glycerol 3-phosphate by adenosine 5-triphosphate (ATP) in a reaction catalysed by glycerok
12、inase (GK): glycerol + ATP GKL-glycerol 3-phosphate + ADP The adenosine 5-diphosphate (ADP) produced is converted back into ATP by phosphoenol pyruvate (PEP) with the formation of pyruvate in a reaction catalysed by pyruvate kinase (PK): ADP + PEP PKATP + pyruvate The pyruvate formed is then convert
13、ed into L-lactate by hydrogenation with nicotinamide adenine dinucleotide (NADH) as reducing agent in a reaction catalysed by the enzyme L-lactate dehydrogenase (L-LDH), the NADH being oxidized to NAD+: pyruvate + NADH + H+LDH-LL-lactate + NAD+The amount of NADH converted by this reaction is equival
14、ent to the amount of glycerol and is determined spectrophotometrically. 5 Reagents 5.1 General Unless otherwise specified, only analytically pure reagents shall be used in the analysis and the water used shall be at least of ISO 3696 grade 3. The following reagents shall be used. 5.2 Doubly distille
15、d water. 5.3 Carrez solution no. 1, prepared by dissolving 3,60 g of potassium hexacyanoferrate(II), K4Fe(CN)6 3 H2O, in water and making the solution up to 100 ml. 5.4 Carrez solution no. 2, prepared by dissolving 7,2 g of zinc sulfate, ZnSO4 7 H2O, in water and making the solution up to 100 ml. 5.
16、5 0,1 mol/l sodium hydroxide solution, NaOH. 5.6 0,5 mol/l sodium hydroxide solution, NaOH. 5.7 Buffer solution, having a pH value of 7,6 and containing 0,75 mol/l of glycerol and 100 mmol/l of Mg2+, prepared by dissolving 10,0 g of glycylglycine and 0,25 g of magnesium sulfate, MgSO4 7 H2O, in 80 m
17、l of water, adjusting the pH value to 7,6 with 2,4 ml of sodium hydroxide (as in subclause 5.6) and making the solution up to 100 ml with water. If stored at +4 C, the solution will be stable for at least three months. DIN 10763:2004-06 5 5.8 NADH/ATP/PEP solution, containing 8,2 mmol/l of nicotinam
18、ide adenine dinucleotide (NADH), 33 mmol/l of adenosine 5-triphosphate (ATP) and 46 mmol/l of phosphoenol pyruvate solution (PEP), prepared by dissolving 42 mg of NADH-Na2, 120 mg of ATP-Na2H2, 60 mg of PEP-Na and 300 mg of sodium hydrogencarbonate (NaHCO3) in 6 ml of water. If stored at +4 C, the s
19、olution will be stable for at least two weeks. 5.9 Coenzyme/buffer mixture, prepared by diluting the NADH/ATP/PEP solution as in subclause 5.8 with the buffer solution as in subclause 5.7 in a ratio of 1:10 before analysis. Use 1,0 ml of this solution for the test. 5.10 Pyruvate kinase/lactate dehyd
20、rogenase suspension, PK/LDH, prepared from 3 mg/ml of pyruvate kinase and 1 mg/ml of lactate dehydrogenase. If stored at +4 C, the suspension will be stable for at least two weeks. 5.11 1 mg/ml glycerokinase suspension, GK. If stored at +4 C, the suspension will be stable for at least one year. NOTE
21、 Commercially available ready-to-use reagents may be used, provided the pipetting procedures and volumes specified by the manufacturer are observed. 6 Apparatus 6.1 General In addition to standard laboratory equipment, the apparatus specified in subclauses 6.2 to 6.13 shall be used. 6.2 Stainless-st
22、eel screen, of aperture size 0,5 mm. 6.3 Beaker, of nominal capacity 100 ml. 6.4 Conical flask, with ground stopper, of nominal capacity 250 ml. 6.5 Enzyme assay pipettes, of nominal capacity 0,01 ml, 1,0 ml, 2,0 ml (e.g. as in DIN 12699) or optionally a piston-operated pipette. 6.6 Fluted filter. 6
23、.7 Funnel, of nominal diameter 70 mm. 6.8 Glass cells, having a path length of 10 mm. Commercially available disposable cells are also suitable. 6.9 Plastic stirrer, for mixing the solutions in cells. 6.10 Graduated pipette, of nominal capacity 1,0 ml. 6.11 Volumetric flask, of nominal capacity 100
24、ml. 6.12 Spectrophotometer, suitable for measuring at a wavelength of 340 nm or, alternatively, a spectral-line photometer fitted with a mercury vapour lamp and suitable for measuring at Hg 334 nm or Hg 365 nm. 6.13 Analytical balance. DIN 10763:2004-06 6 7 Procedure 7.1 Sample preparation 7.1.1 Pur
25、e liquid or set honey Adequately homogenize the laboratory sample by stirring it vigorously for not less than three minutes, taking care to minimize the amount of air included. 7.1.2 Impure liquid or set honey After removing coarse impurities, stir the honey at ambient temperature until smooth and p
26、ass it through a screen (as in subclause 6.2), using a spatula in the case of set honey. 7.1.3 Comb honey De-cap the combs if still capped, then separate the honey completely from the combs without heating, using a screen (as in subclause 6.2). 7.2 Preparation of sample solution Weigh about 20 g of
27、the sample prepared as in subclause 7.1 to an accuracy of 1 mg into a 100 ml beaker or a 100 ml conical flask and add about 60 ml of water while stirring. Transfer the content of the beaker or the conical flask quantitatively to a 100 ml volumetric flask, completing the transfers by using water to r
28、inse out the container. Then add 1,0 ml of Carrez solution no. 1 (as in subclause 5.3) followed by 1,0 ml of Carrez solution no. 2 (as in subclause 5.4). Mix thoroughly and make the contents of the flask up to the mark. NOTE If cloudiness occurs, neutralize the solution with sodium hydroxide solutio
29、n (as in subclause 5.5). Filter the contents of the flask through a fluted filter (as in subclause 6.6) into a conical flask (as in subclause 6.4). Use the filtrate (sample solution) for the glycerol determination (as in subclause 7.3). 7.3 Preparation of test solutions and determination React the b
30、lank solution and the test solution using the pipetting procedure shown in table 1 and perform the analysis under the following conditions: Wavelength 340 nm, Hg 365 nm or Hg 334 nm Cell 1,00 cm path length Temperature 20 C to 25 C Test volume 3,120 ml Determination against air (no cell in beam path
31、), or water Sample solution 1 g to 40 g of glycerol per cell (in 0,100 ml to 2,000 ml sample volume) Differential absorbance, E 0,1 to 1,0 DIN 10763:2004-06 7 Table 1 Pipetting procedure Pipette into the cells Blank, in ml Test solution, in ml Coenzyme/buffer mixture (5.9) 1,0 1,0 Sample solution (7
32、.2) 0,10 Before adding the sample solution rinse the enzyme assay pipette or the tip of the piston-operated pipette with sample solution. Water (5.2) 2,00 1,90 PK/LDH suspension (5.10) 0,01 0,01 Mix e.g. with a stirring spatula or by swirling after sealing with laboratory foil. Determine the absorba
33、nce of the solution (E1) after about three minutes and then start the reaction by adding: Glycerokinase suspension (5.11) 0,01 0,01 Mix the solution e.g. with a stirring spatula or by swirling after sealing the cell with laboratory foil. Wait for the reaction to stop (in about 5 to 10 min), then det
34、ermine the absorbances of the solutions immediately after one another (E2). If the reaction has not stopped after 15 min, continue to determine the absorbances at 2 min intervals until a constant decrease in absorbance every 2 min is reached. If constant decreases in absorbance are found for E2, ext
35、rapolate the absorbances to the time the glycerokinase suspension was added. 8 Calculation Calculate the differential absorbance, E, from the difference in absorbance between the sample (test solution) and the blank using equation (1): E = (E1 E2)test solution (E1 E2)blank(1) To obtain a sufficientl
36、y accurate result, the measured differential absorbance, E, shall generally be at least 0,100 absorbance units. If the differential absorbance of the sample (Esampleand the sample solution shall be diluted. Calculate the concentration by mass for which the amount of substrate is equivalent to twice
37、the amount of NADH using equation (2): EVdMV=00010001PT (2) where is the concentration by mass of glycerol, in mg/l; VTis the test volume, in ml; VPis the sample volume, in ml; M is the molar mass of glycerol (92,1 g); ) is greater than 1 000 (if determined at 340 nm or 334 nm (Hg) or 0,500 (if dete
38、rmined at 365 nm (Hg), the concentration of glycerol in the solution is too high ,DIN 10763:2004-06 8 d is the path length of the cell, in cm (here: 1,00 cm); is the absorptivity of NADH (6,3 l mmol1 cm1at 340 nm, 6,18 l mmol1 cm1at Hg 334 nm and 3,4 l mmol1 cm1at Hg 365 nm); E is the differential a
39、bsorption given by equation (1). Calculate the concentration by mass of glycerol in the sample solution using equation (3): EE =78120001100000100011920203,(3) where is the concentration by mass of glycerol in the sample solution, in mg/l; is the absorptivity of NADH (see above); E is the differentia
40、l absorbance given by equation (1). If the sample solution has been diluted during preparation, multiply the result by the dilution factor, F. Calculate the proportion by mass, w, of glycerol, in mg/kg, using equation (4): 0001=mw(4) where is the concentration by mass of glycerol in the sample solut
41、ion, in mg/l; m is the initial mass of the sample, in g/l. Above 10 mg/kg, report the result to three significant places. 9 Reliability 9.1 Specificity The determination is specific for glycerol. Dehydroxyacetone is not converted under the specified conditions. If pure glycerol is analysed, results
42、of about 100 % relative to the anhydrous substance are to be expected. 9.2 Sensitivity and limit of detection The smallest differential absorbance the method can detect is 0,005 absorbance units. For a maximum sample volume, VP, of 2,000 ml and determination at 340 nm, this corresponds to a glycerol
43、 concentration of 0,1 mg/l (or 2 mg/l of sample solution for a sample volume, VP, of 0,100 ml). The differential absorbance of 0,020 and the maximum sample volume, VP, of 2,000 ml result in a limit of detection of 0,4 mg/l (determined at 340 nm). DIN 10763:2004-06 9 9.3 Linearity Linearity exists fr
44、om about 1 g of glycerol per assay (0,4 mg of glycerol per litre of sample solution, with a sample volume, VP, of 2,000 ml) to 40 g of glycerol per assay (0,4 g of glycerol per litre of sample solution, with a sample volume, VP, of 0,100 ml). 10 Precision 10.1 General The precision data have been de
45、termined in an interlaboratory test on four honeys having different glycerol contents, performed as specified in ISO 5725-1 and ISO 5725-2. The results will be found in Annex A. 10.2 Repeatability limit (same operator, same equipment) The absolute difference between two successive results obtained u
46、nder repeatability conditions will not exceed the repeatability limit, r, in more than 5 % of cases. Values of r and the repeatability standard deviation, sr, are given in the annex. 10.3 Reproducibility limit (different operators, different equipment) The absolute difference between two individual
47、results obtained under reproducibility conditions will not exceed the reproducibility limit, R, in more than 5 % of cases. Values of R and of the reproducibility standard deviation, sR, are given in the annex. 11 Test report The test report shall include the following information as a minimum: a) al
48、l the information necessary to identify the sample, e.g. the type of sample, its origin and description; b) a reference to this standard/the method used; c) glycerol content, in mg/kg of honey; d) type and date of sampling (if known); e) date of receipt of sample; f) analysis date; g) any unusual de
49、tails observed during the analysis; h) any operations undertaken optionally and not included in this standard that may have influenced the result. DIN 10763:2004-06 10 Annex A (informative) Results of interlaboratory testing The Technical Committee Honiguntersuchung performed an interlaboratory test and evaluated it as specified in ISO 5725-1 and ISO 5725-2. The statistical results are shown in table A.1. Table A.1 Results of the interlaboratory test (values in mg/kg) Parameter
