1、ICS 65.160Tabak und Tabakerzeugnisse Bestimmung von Konservierungsstoffen mit Hochleistungsflssig-chromatographieIn keeping with current practice in standards published by the International Organization for Standardization(ISO), a comma has been used throughout as the decimal marker.Ref. No. DIN 103
2、77 : 2003-10English price group 11 Sales No. 011110.04DEUTSCHE NORM October 2003 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 exclusive right of sale for German Standards
3、(DIN-Normen).Translation by DIN-Sprachendienst.In case of doubt, the German-language original should be consulted as the authoritative text.Determination of preservatives in tobacco andtobacco products using high-performanceliquid chromatographyContinued on pages 2 to 13.10377ForewordThis standard h
4、as been prepared by Technical Committee Tabak- und Tabakrauchanalyse of the Normen-ausschuss Lebensmittel und landwirtschaftliche Produkte (Foodstuffs and Agricultural Products StandardsCommittee).1 ScopeThis standard specifies a method of determining benzoic acid, sorbic acid, methyl p-hydroxybenzo
5、ate, ethylp-hydroxybenzoate and propyl p-hydroxybenzoate as preservatives in tobacco and tobacco products usinghigh-performance liquid chromatography. The method has been found to be suitable for concentrations of100 mg per kg to 2 000 mg per kg of sorbic acid and of 100 mg per kg to 5 000 mg per kg
6、 of benzoic acidand p-hydroxybenzoates.ContentsPageForeword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 Norm
7、ative references . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 Principle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8、. . . . . . . . . . . . . . . . . . . . . . 25 Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Procedure . .
9、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Expression of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10、. . . . . . . . . . . . . . . . . . 410 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6Annex A Example of suitable HPLC parameters and of chromatograms . . . . 6Annex B Results of interlaboratory testing . . . . . . . . . . . . . .
11、. . . . . . . . . . . . . . 8Page 2DIN 10377 : 2003-102 Normative referencesThis standard incorporates, by dated or undated reference, provisions from other publications. These norma-tive references are cited at the appropriate places in the text, and the titles of the publications are listed below.
12、For dated references, subsequent amendments to or revisions of any of these publications apply to thisstandard only when incorporated in it by amendment or revision. For undated references, the latest edition ofthe publication referred to applies.DIN 10252 Determining the water content of tobacco an
13、d tobacco products by the Karl FischermethodDIN 10257-1 Sampling of fine-cut tobacco GeneralISO 3696 : 1987 Water for analytical laboratory use Specification and test methodsISO 4874 : 1981 Tobacco Sampling of batches of raw material General principlesISO 5725-1 : 1994 Accuracy (trueness and precisi
14、on) 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 determination of repeatability and reproducibility of standard measurementmethod (including Technical Corrige
15、ndum 1 : 2002)ISO 8243 : 1991 Cigarettes Sampling3 PrincipleThe preservatives are extracted using a methanol/water mixture. After the extract has been filtered, they areseparated by high-performance liquid chromatography (HPLC) with UV or diode array detection, identified bytheir retention times and
16、 determined quantitatively from the peak areas using external standards.4 Reagents4.1 GeneralUnless otherwise specified, analytical grade reagents and water of grade 3 as in ISO 3696 shall be used.4.2 10% (V/V) acetic acid, CH3COOH.4.3 Methanol, suitable for HPLC.4.4 0,01 mol/l ammonium acetate solu
17、tion, CH3COONH4.4.5 Extraction solution, prepared from 70 parts by volume of methanol and 30 parts by volume of water.4.6 HPLC mobile phase for gradient elution, consisting of ammonium acetate solution and methanol. ThepH of mobile phase A shall be adjusted to 4 to 4,5 with acetic acid to ensure tha
18、t the vanillin and benzoic acidare separated. Examples of mobile phase A are given in Annex A.4.7 Reagents used for calibration4.7.1 Benzoic acid.4.7.2 Sorbic acid.4.7.3 Methyl p-hydroxybenzoate (methyl pHB).4.7.4 Ethyl p-hydroxybenzoate (ethyl pHB).4.7.5 Propyl p-hydroxybenzoate (propyl pHB).4.8 St
19、ock solution, containing 1 000 mg/l of each calibration reagent as in subclause 4.7, prepared byweighing 100 mg of each reagent into a 100 ml volumetric flask, dissolving in 70 ml of methanol and makingup to 100 ml with water. If stored in a refrigerator, the stock solution will be stable for at lea
20、st four weeks.4.9 Calibration solutionsPrepare the calibration solutions listed in table 1 from the stock solution by dilution with the extraction solution.Page 3DIN 10377 : 2003-10Table 1: Calibration solutionsDilution ratio of stock solution Concentration, in mg/l1) Concentration, in mg/kg2)1 :1 0
21、00 1 501 : 500 2 1001 : 100 10 5001 : 50 20 1 0001 : 20 50 2 5001) Milligrams of preservative per litre of calibration solution (for an initial mass of 100 mg in each case).2) Milligrams of preservative per kilogram of tobacco product (for an initial mass of 1 g and 50 mlof extraction solution).Prep
22、are fresh calibration solutions every week. If contents are likely to be low, perform an additional calibrationin the lower range.4.10 Vanillin solution, used as test solution for determining the column efficiency, containing 50 mg/l to100 mg/l of vanillin in the extraction solution.5 ApparatusIn ad
23、dition to standard laboratory equipment, the following shall be used.5.1 Analytical balance, capable of weighing to an accuracy of 0,1 mg.5.2 Injection syringe, for membrane filter.5.3 Membrane filter, having a pore size of 0,45 m or less.5.4 HPLC gradient system, comprising a diode array detector (
24、DAD) or a UV detector, with switchablewavelengths; a column oven is recommended.5.5 HPLC columns, packed with analytical RP-18 reverse phase, capable of clearly separating vanillin andbenzoic acid (see Annex A for examples of chromatograms). It has been found advantageous to use a suitableprecolumn.
25、6 SamplingA representative sample shall be collected. Sampling may also be carried out as specified in DIN 10257-1,ISO 4874 or ISO 8243.7 Procedure7.1 Sample preparationWeigh 1 g of finely ground tobacco to an accuracy of three decimal places and transfer it to a conical flask. Add50 ml of extractio
26、n solution and extract on a shaking machine set to approximately 165 min1for 30 minutes.Alternatively, extract for 20 minutes in an ultrasonic bath.For concentrations below 200 mg/kg, extraction with 20 ml of extraction solution is recommended. If theconcentration found is beyond the calibration ran
27、ge, repeat the determination with a lower initial mass.Filter the extract through a fluted filter paper, discarding the first millilitres of filtrate, and then filter again througha membrane filter. If stored in a refrigerator at 4 C, the test solutions will be stable for three days.7.2 Identificati
28、onIdentify the preservatives to be determined by comparing the retention times of the individual peaks of the testsolution with those of the calibration solutions. Using a DAD may occasionally provide additional informationfor validating positive findings.Examples of suitable HPLC parameters and of
29、chromatograms will be found in Annex A.Page 4DIN 10377 : 2003-107.3 CalibrationPerform a five-point calibration using the calibration solutions. Check the linearity of the calibration functionover the entire measurement range. Duplicate injections of calibration and test solutions are recommended.Ch
30、eck the stability of the analytical system using a suitable calibration solution after injecting approximatelyten samples.7.4 AnalysisAdjust the HPLC system so as to obtain adequate separation of the analytes from the matrix substances,checking, in particular, whether benzoic acid and vanillin are s
31、eparated. Inject suitable volumes, usually 10 lportions, of calibration and test solutions into the HPLC system. Integrate the peak areas and compare theresults with the corresponding values for the calibration substances.8 Expression of resultsDetermine the preservative contents from the peak areas
32、 in a multi-point calibration using external standards.Calculate the contents as a proportion by mass, v, in mg per kg, of sample material, using the followingequation:rKVE 1 000v = (1)mp 1 000whererKis the concentration of the preservative concerned, determined by linear regression, in g per ml ofe
33、xtraction solution;VEis the extract volume, in ml;mpis the initial sample mass, in g.Calculate the contents of the preservative concerned as a proportion by mass, vTM, in mg per kg of dry matter,using the following equation:v 100vTM= (2)100 vH2Owherev is the proportion by mass of the preservative co
34、ncerned, in mg per kg of sample material, calculatedusing equation (1);vH2Ois the water content of the sample material determined as in DIN 10252, as a percentage.Report the contents of the preservatives concerned, in mg per kg of dry matter with no decimal place.9 Precision9.1 GeneralSee Annex B fo
35、r details of an interlaboratory test for determining the precision data.9.2 Repeatability limit(same operator, same equipment)The absolute difference between two successive results obtained under repeatability conditions will not exceedthe repeatability limit, r, in more than 5% of cases.An interlab
36、oratory test yielded the repeatability limits for the determination of individual preservatives in to-bacco products shown in table 2.9.3 Reproducibility limit(different operators, different equipment)The absolute difference between two individual results obtained under reproducibility conditions wi
37、ll notexceed the reproducibility limit, R, in more than 5% of cases.An interlaboratory test yielded the reproducibility limits for the determination of individual preservatives intobacco products shown in table 3.Page 5DIN 10377 : 2003-10Table 2: Repeatability limitsPreservativeRepeatability limits,
38、 r, in g per 100 g of dry matter1)Tobacco A Tobacco B Tobacco C Tobacco D Tobacco ESorbic acid 0,007 5 0,002 6 0,000 4Benzoic acid 0,002 9 0,021 3 0,005 7 0,003 6Methyl pHB 0,023 4 0,004 7 0,001 0Ethyl pHB 0,023 3 0,003 6 0,000 5Propyl pHB 0,023 7 0,003 5 0,001 2Matrix:Tobacco A: raw tobacco, withou
39、t added preservatives.Tobacco B: commercial pipe tobacco, without added preservatives.Tobacco C: commercial pipe tobacco, with 2 000 mg/kg of sorbic acid, 5 000 mg/kg of benzoic acid and5 000 mg/kg each of methyl pHB, ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).Tobacco D:
40、commercial pipe tobacco, with 1 000 mg/kg of sorbic acid, 1 000 mg/kg of benzoic acid and1 000 mg/kg each of methyl pHB, ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).Tobacco E: commercial pipe tobacco, with 100 mg/kg sorbic acid, 100 mg/kg benzoic acid and 100 mg/kgeach of
41、methyl pHB, ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).1) The values are reported with outliers eliminated, all the data obtained by one laboratory being excludedfrom the evaluation for this reason.Table 3: Reproducibility limitsPreservativeReproducibility limits, in g pe
42、r 100 g of dry matter1)Tobacco A Tobacco B Tobacco C Tobacco D Tobacco ESorbic acid 0,018 1 0,008 3 0,007 1Benzoic acid 0,009 1 0,054 3 0,016 8 0,010 6Methyl pHB 0,097 8 0,021 4 0,004 8Ethyl pHB 0,091 7 0,018 9 0,003 1Propyl pHB 0,067 2 0,011 9 0,003 0Matrix:Tobacco A: raw tobacco, without added pre
43、servatives.Tobacco B: commercial pipe tobacco, without added preservatives.Tobacco C: commercial pipe tobacco, with 2 000 mg/kg of sorbic acid, 5 000 mg/kg of benzoic acid and5 000 mg/kg each of methyl pHB, ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).Tobacco D: commercial
44、pipe tobacco, with 1 000 mg/kg of sorbic acid, 1 000 mg/kg of benzoic acid and1 000 mg/kg each of methyl pHB, ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).Tobacco E: commercial pipe tobacco, with 100 mg/kg sorbic acid, 100 mg/kg benzoic acid and 100 mg/kgeach of methyl pHB,
45、 ethyl pHB and propyl pHB added (all esters calculated as benzoic acid).1) The values are reported with outliers eliminated, all the data obtained by one laboratory being excludedfrom the evaluation for this reason.Page 6DIN 10377 : 2003-1010 Test reportThe test report shall refer to this standard a
46、nd include the following details:a) identification of sample (type, origin, description);b) date of sampling and method (if known);c) date of delivery of sample;d) date of testing;e) test results and units in which they are reported;f) any peculiarities observed during the test;g) any deviation from
47、 this method that may have affected the results.Annex AExample of suitable HPLC parameters and of chromatogramsA.1 Example 1A.1.1 ODS HPLC parametersStationary phase: ODS AQ1)Column dimensions: 250 mm 4mmMobile phase A: (75 + 25) (V/V) ammonium acetate solution and methanolMobile phase B: methanolFl
48、ow rate: 0,7 ml/minVolume injected: 10 lOven temperature: 40 C or 50 C, depending on column efficiencyDetection: UV with a wavelength of 227 nm for benzoic acid and 260 nm for sorbic acid andpHBsGradient (see table below)Mobile phase BTime,Concentration,in minutesas a percentage by volume00,0 021,0
49、022,0 1530,0 4540,0 4540,1 0Equilibration: 10 min 01) Reference toODS AQis for the purpose of providing the user with an example of a suitable commercialproduct and does not imply any approval by DIN.Page 7DIN 10377 : 2003-10A.1.2 ChromatogramVanillin - 10,525Benzoic acid - 11,482Sorbic acid - 17,726Methyl pHB - 26,083Ethyl pHB - 32,875Propyl pHB - 37,006Figure A.1: Separation of a 50 g/ml standard solution using ODS AQ 1)A.2 Example 2A.2.1 Lichrospher 2) HPLC parametersStationary phase: LichrospherColumn dimensions:
