1、 DEUTSCHE NORM June 2004DIN 10762 ICS 67.180.10 Analysis of honey Determination of ethanol content Enzymatic method Untersuchung von Honig Bestimmung des Gehaltes an Ethanol Enzymatisches Verfahren Document comprises 11 pagesTranslation by DIN-Sprachendienst. In case of doubt, the German-language or
2、iginal 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 price
3、group 9 www.din.de www.beuth.de !,in“05.06 9707529DIN 10762:2004-06 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 .5 8 Calculation7 9 Reliability of method .8 10 Precision.9 11 Test report 9 Annex A (infor
4、mative) Results of interlaboratory testing 10 Bibliography. 11 DIN 10762:2004-06 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 Scop
5、e This standard specifies an enzymatic method of determining the ethanol 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
6、of the publications 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 an
7、y amendments). 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 Pa
8、rt 1: General 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, t
9、he following terms and definitions apply. 3.1 ethanol content the ethanol content of honey determined by the method described in this standard and reported in mg/kg of sample material 3.2 specificity the ability to detect the analyte and distinguish it from similar substances, impurities and degrada
10、tion 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 10762:2004-06 4 3.5 li
11、nearity 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 Ethanol is oxidized to acetaldehyde by nicotinamide adenine dinucleotide (NAD+) in the following reaction, which is catalysed by the enzyme a
12、lcohol dehydrogenase (ADH): ethanol + NAD+ ADHacetaldehyde + NADH + H+However the equilibrium of this reaction is on the ethanol and NAD side and can be displaced to the right-hand side in an alkaline medium by oxidizing the acetaldehyde quantitatively to acetic acid in a reaction catalysed by aldeh
13、yde dehydrogenase (Al-DH): acetaldehyde + NAD+ H2O DH-Alacetic acid + NADH + H+The NADH formed is equivalent to the amount of ethanol and is determined spectrophotometrically. 5 Reagents 5.1 General Unless otherwise specified, only analytically pure reagents shall be used in the analysis and the wat
14、er used shall be at least of ISO 3696 grade 3. The following reagents shall be used. 5.2 Doubly distilled 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, prep
15、ared by dissolving 7,20 g of zinc sulfate, ZnSO4 7 H2O, in water and making the solution up to 100 ml. 5.5 0,1 mol/l sodium hydroxide solution, NaOH. 5.6 Diphosphate buffer solution, prepared by dissolving 3,33 g of potassium diphosphate, K4P2O7, in 60 ml of water, adjusting the pH value to 9 with 1
16、 mol/l hydrochloric acid and making the solution up to 100 ml with water. If stored at +4 C, the solution will be stable for at least two months. 5.7 98 % (m/m) -nicotinamide adenine dinucleotide, -NAD, as free acid. 5.8 Aldehyde dehydrogenase, Al-DH, freeze dried, from yeast, having an enzyme activ
17、ity of about 15 U/mg1). If stored dry at +4 C, it will be stable for at least six months. 1) In this standard, enzyme activity is specified in international units (U), where 1 U is the enzyme activity that converts 1 mol of substrate per minute under optimum conditions at 25 C. DIN 10762:2004-06 5 5
18、.9 Reaction mixture, prepared by dissolving 114 mg of -NAD (as in subclause 5.7) and 3,33 mg of Al-DH (as in subclause 5.8) having an enzyme activity of about 50 U in 100 ml of diphosphate buffer solution while stirring. If stored at +4 C, the solution will be stable for one day. 5.10 Alcohol dehydr
19、ogenase suspension, ADH, from yeast, having a concentration by mass, ADH, of 30 mg/ml in 3,2 mol/l ammonium sulfate solution, (NH4)2SO4, and having an enzyme activity of about 9 000 U/ml1). If stored at +4 C, the alcohol dehydrogenase solution will be stable for at least four months. NOTE Ready-to-u
20、se reagents are commercially available. If they are used, the pipetting procedure and volumes specified by the manufacturer shall be observed. 6 Apparatus 6.1 General In addition to standard laboratory equipment, the apparatus specified in subclauses 6.2 to 6.12 shall be used. 6.2 Stainless-steel sc
21、reen, of aperture size 0,5 mm. 6.3 Beaker, of nominal capacity 100 ml, or conical flask, 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 0,05 ml, 0,5 ml, 3,0 ml (e.g. as in DIN 12699) or optionally piston-
22、operated pipettes. 6.6 Fluted filter. 6.7 Funnel, of nominal diameter 70 mm. 6.8 Glass cells, with 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 Volumetric flask, of nominal capacity 100 ml. 6.11 Spectr
23、ophotometer, or, alternatively, a spectral-line photometer fitted with a mercury vapour lamp. 6.12 Analytical balance. 7 Procedure 7.1 Sample preparation 7.1.1 Pure liquid or set honey Adequately homogenize the laboratory sample by stirring it vigorously for not less than three minutes, taking care
24、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 pass it through a screen (as in subclause 6.2), using a spatula in the case of set honey. DIN 10762:2004-06 6 7.1.3 Comb honey De-cap the
25、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 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 (as in
26、subclause 6.3) 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 transfer by rinsing the container with water. Then add 1,0 ml of Carrez solution no. 1 (as in subclause 5.3) followed by 1,0
27、 ml 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 using sodium hydroxide solution (as in subclause 5.5). Filter the contents of the flask through a fluted filter (as in subclause 6.6)
28、into a conical flask (as in subclause 6.4). Use the filtrate (sample solution) for the ethanol (determination as in subclause 7.3). 7.3 Preparation of test solutions and determination React the blank solution and the test solution (as in subclause 7.2) using the pipetting procedure shown in table 1
29、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: 3,55 ml Determination against air (no cell in beam path), water or blank Sample solution: 0,3 g to 12 g of ethanol per cell (in 0,1 ml
30、to 0,5 ml sample volume) Seal the cells during the determination, e.g. with laboratory foil. Troublesome bubble formation at the inside wall of the cell can be cured by sharply tapping the cell or by briefly dipping one corner of the cell into an ultrasonic bath. Because ethanol is very volatile, th
31、e following measures shall be adopted in handling the sample, in preparing dilutions and when pipetting the sample solution into the cell: a) the filtrate shall not be added to the collecting vessel in drops but shall be allowed to run down its walls; b) when alcohol-containing solutions are dispens
32、ed, they shall always be pipetted underneath the surface of the water (when preparing dilutions) or of the buffer (in the case of the mixture used for analysis); c) when pipetting very dilute sample solutions into the test solution, the enzyme assay pipette (as in subclause 6.5) shall be rinsed at l
33、east six times beforehand and the pipette tip of a piston-operated pipette (as in subclause 6.5) shall be rinsed at least three times beforehand; d) the same piston-operated pipette shall never be used for diluting alcohol-containing samples and for adding reagents; e) the atmosphere and the water s
34、hall both be alcohol-free. DIN 10762:2004-06 7 Table 1 Pipetting procedure Pipette into the cells Blank, in ml Test solution, in ml Reaction mixture (as in 5.9) 3,00 3,00 Water (as in 5.2) 0,50 Sample solution (as in 7.2) 0,50 After sealing with laboratory foil, mix using e.g. a stirring spatula or
35、by swirling. Before adding the sample solution rinse the enzyme assay pipette or the tip of the piston-operated pipette with sample solution. After about three minutes measure the absorbance (E1) and then start the reaction by adding: Alcohol dehydrogenase solution (as in 5.10) 0,05 0,05 Mix the sol
36、ution and, after about 5 to 10 minutes measure the absorbances of the solutions immediately after one another (E2). The amount of ethanol in the sample solution shall be between 0,5 g and 12 g (for measurement at 365 nm) or 0,3 g and 6 g (for measurement at 340 or 334 nm). To achieve a sufficiently
37、large difference in absorbance, dilute the sample solution until the ethanol concentration is between 0,02 g/l and 0,12 g/l or 0,01 g/l and 0,06 g/l, respectively. 8 Calculation Calculate the differential absorbance, E, from the difference in absorbance between the sample (test solution) and the bla
38、nk using equation (1): E = (E2 E1)test solution (E2 E1)blank(1) To obtain a sufficiently accurate result, the measured differential absorbance E shall generally be at least 0,100 absorbance units. Calculate the concentration by mass for which the amount of substrate is equivalent to twice the amount
39、 of NADH using equation (2): EVdMV=000120001PT (2) where is the concentration by mass of ethanol, in mg/l; VTis the test volume, in ml; VPis the sample volume, in ml; M is the molar mass of ethanol (46,07 g); d is the path length of the cell, in cm (here: 1,00 cm); is the absorptivity of NADH (6,3 l
40、 mmol1 cm1at 340 nm, 6,18 l mmol1 cm1at Hg 334 nm and 3,4 l mmol1 cm1at Hg 365 nm). DIN 10762:2004-06 8 Calculate the concentration by mass of ethanol in the sample solution using equation (3): EE =516300012500000100010746553 ,(3) where is the concentration by mass of ethanol in the sample solution,
41、 in mg/l; is the absorptivity of NADH (see above); E is the differential 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 ethanol, in mg/kg, using equation (4): 0001=mw
42、(4) where is the concentration by mass of ethanol in the sample solution, 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 of method 9.1 Specificity Any interference due to aldehydes and ketones is eliminated by the or
43、der in which the reagents are added. Methanol is not converted due to the unfavourable KMvalues2)of the enzymes used. n-propanol and n-butanol are quantitatively converted under the test conditions, but higher primary alcohols result in a sample-dependent creep reaction. Secondary, tertiary and arom
44、atic alcohols do not react and glycerol does not interfere with the test even at fairly high concentrations. Results of about 100 % are to be expected if pure ethanol is analysed. NOTE A recovery of less than 100 % does not necessarily mean incomplete conversion in the enzymatic determination, but i
45、s more likely to indicate the loss of analyte during handling, preparing the ethanol solution and pipetting the dilute ethanol solution into the test solution. 9.2 Sensitivity and limit of detection The smallest differential absorbance the method can detect is 0,005 absorbance units. For a maximum s
46、ample volume, VP, of 0,500 ml, a test volume, VT, of 3,550 ml and determination at 340 nm, this corresponds to an ethanol concentration of about 0,1 mg/l (or 0,6 mg/l of sample solution for a sample volume, VP, of 0,100 ml and a test volume, VT, of 3,15 ml). 2) The Michaelis constant, KM, is defined
47、 as the substrate concentration at which the reaction rate reaches max/2. DIN 10762:2004-06 9 The differential absorbance of 0,020, the maximum sample volume, VP, of 0,500 ml and the test volume, VT, of 3,550 ml result in a limit of detection of 0,5 mg/l. 9.3 Linearity Linearity exists from about 0,
48、3 g of ethanol per assay (0,5 mg of ethanol per litre of sample solution, with a sample volume, VP, of 0,5 ml and a test volume, VT, of 3,550 ml) to 12 g of ethanol per assay (0,12 g of ethanol per litre of sample solution, with a sample volume, VP, of 0,100 ml and a test volume, VT, of 3,150 ml). 1
49、0 Precision 10.1 General The precision data have been determined in an interlaboratory test on three honeys having different ethanol 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 under repeatability conditions will not exceed the repeatability limit, r, in more than 5 % of cases. Values of r and
