1、BS ISO20369:2009ICS 65.160NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMaterial used forproducing wrappingsfor cigarette filters,cigarettes and othertobacco products Determination ofcitrate contentThis British Standardwas published under theauthority of the S
2、tandardsPolicy and StrategyCommittee on 31 October2009 BSI 2009ISBN 978 0 580 63201 3Amendments/corrigenda issued since publicationDate CommentsBS ISO 20369:2009National forewordThis British Standard is the UK implementation of ISO 20369:2009.The UK participation in its preparation was entrusted to
3、TechnicalCommittee AW/40, Tobacco and tobacco products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance
4、 with a British Standard cannot confer immunityfrom legal obligations.BS ISO 20369:2009Reference numberISO 20369:2009(E)ISO 2009INTERNATIONAL STANDARD ISO20369First edition2009-10-01Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of ci
5、trate content Matriaux utiliss pour la fabrication des enveloppes pour les filtres de cigarette, pour les cigarettes et pour les autres produits du tabac Dosage du citrate BS ISO 20369:2009ISO 20369:2009(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licens
6、ing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobes licensing policy. The ISO
7、Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been
8、 taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO 2009 All rights reserved. Unless otherwise specified, no part of t
9、his publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISOs member body in the country of the requester. ISO copyright office Case postale 56 CH-1
10、211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyrightiso.org Web www.iso.org Published in Switzerland ii ISO 2009 All rights reservedBS ISO 20369:2009ISO 20369:2009(E) ISO 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwi
11、de federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on tha
12、t committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in a
13、ccordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard re
14、quires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 20369 was prepared by Techn
15、ical Committee ISO/TC 126, Tobacco and tobacco products. BS ISO 20369:2009BS ISO 20369:2009INTERNATIONAL STANDARD ISO 20369:2009(E) ISO 2009 All rights reserved 1Material used for producing wrappings for cigarette filters, cigarettes and other tobacco products Determination of citrate content WARNIN
16、G The use of this International Standard can involve hazardous materials, operations and equipment. This International Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this International Standard to establish appropriate sa
17、fety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard specifies a method for the determination of the citrate content of material used to produce wrappings for cigarette filters, cigarettes and other tobacco products. 2
18、Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 187, Paper, boards and p
19、ulps Standard atmosphere for conditioning and testing and procedure for monitoring the atmosphere and conditioning of samples ISO 287, Paper and board Determination of moisture content of a lot Oven-drying method ISO 3696, Water for analytical laboratory use Specification and test methods 3 Terms an
20、d definitions For the purposes of this document, the following terms and definitions apply. 3.1 citrate content materials for producing wrappings for cigarette filters, cigarettes and other tobacco products anhydrous citric acid content determined by the enzymatic method NOTE Citrate is generally ad
21、ded to wrapping materials, in particular cigarette paper, as trisodium citrate and tripotassium citrate or mixtures thereof to influence the burning rate of the cigarette and, consequently, the puff number. BS ISO 20369:2009ISO 20369:2009(E) 2 ISO 2009 All rights reserved4 Principle The citrate cont
22、ent is determined by an enzymatic method in which (citrate) citric acid, catalyzed by the enzyme citrate lyase (CL), is first converted to oxaloacetate and acetate in accordance with the following reaction: citrate CLoxaloacetate + acetate (1) In the presence of the enzymes L-malate dehydrogenase (L
23、-MDH) and L-lactate dehydrogenase (L-LDH), oxaloacetate and its decarboxylation product, pyruvate, are converted by reduced nicotinamide adenine dinucleotide (NADH) to L-malate and L-lactate, respectively, according to the following reactions: oxaloacetate + NADH + H+L-MDHL-malate + NAD+(2) pyruvate
24、 + NADH + H+L-LDHL-lactate + NAD+(3The amount of oxidized NADH is proportional to the amount of citrate. The residual NADH is determined from its absorbance at 340 nm by spectrophotometry. 5 Reagents 5.1 General All reagents used shall be of recognized analytical grade. Water used shall be in accord
25、ance with at least grade 3 of ISO 3696. 5.2 Test kit for enzymatic citrate determination 5.2.1 General Commercially available test kits shall be used that generally contain two reagent mixtures Roche-Biopharm 10.139.076.035, or equivalent1). Optionally, the determination may be performed using indiv
26、idual reagents. In that case, the procedure is to be found in the literature or commercial information documents. 5.2.2 Reagent mixture 1 Reagent mixture 1 shall be diluted with water in accordance with the manufacturers instructions to produce solution 1. The ready-to-use solution 1, which is buffe
27、red to a pH of 7,8 using glycylglycine buffer, contains the following: L-malate dehydrogenase (L-MDH), about 12 IU2)/ml; L-lactate dehydrogenase (L-LDH), about 23 IU/ml; 1) Roche-Biopharm 10.139.076.035 is an example of a suitable product available commercially. This information is given for the con
28、venience of the users of this International Standard and does not constitute an endorsement by ISO of this product. 2) IU (international unit) is the amount of enzyme (activity) that catalyses the conversion of 1 mol of substrate per minute under standard conditions. BS ISO 20369:2009ISO 20369:2009(
29、E) ISO 2009 All rights reserved 3 reduced nicotinamide adenine dinucleotide (NADH), 0,5 mg/ml; stabilizers. Solution 1 may be stored for two weeks at +4 C or for four weeks at 20 C. 5.2.3 Reagent mixture 2 Reagent mixture 2 shall be diluted with water in accordance with the manufacturers instruction
30、s to produce solution 2. The ready-to-use solution 2 contains about 40 IU/ml of citrate lyase. Solution 2 may be stored for two weeks at +4 C or for four weeks at 20 C. The activity of the enzyme system shall be (100 5) %. 5.3 Citric acid monohydrate. 6 Apparatus Usual laboratory apparatus and, in p
31、articular, the following items. 6.1 Conical flasks, of nominal capacity 250 ml. 6.2 Funnel, of diameter 80 mm. 6.3 Filter paper, of diameter 125 mm Whatman No. 40, or equivalent3). 6.4 Pipettes, with graduations suitable for nominal capacities of 1 ml, 2 ml, 5 ml and 10 ml; enzyme assay pipettes mig
32、ht be used as well 2. 6.5 Piston-operated pipette, of nominal capacity 20 l. 6.6 Double-beam spectrophotometer, suitable for a wavelength of 340 nm. 6.7 Glass or plastic cuvets, of light path 10 mm and capacity 5 ml. 6.8 Ultrasonic bath or magnetic stirrer. 6.9 Analytical balance, suitable for measu
33、ring to the nearest 0,001 g. 7 Procedure 7.1 Sample preparation Extract approximately 1,0 g, to the nearest 0,001 g, of cut wrapping material previously conditioned as specified in ISO 187, in 100 ml of water in a 250 ml conical flask (6.1), by the aid of an ultrasonic bath or magnetic stirrer (6.8)
34、 for 30 min. Then filter the extract through a filter paper (6.3). 7.2 Determination Perform the determination at a constant temperature of between 20 C and 25 C. The following pipetting procedure (see Table 1) has proved satisfactory for the blank solution (water) and the test solution (sample extr
35、act as prepared in 7.1). 3) Whatman No. 40 is an example of a suitable product available commercially. This information is given for the convenience of the users of this International Standard and does not constitute an endorsement by ISO of this product. BS ISO 20369:2009ISO 20369:2009(E) 4 ISO 200
36、9 All rights reservedThe absorbance shall be determined using a double-beam spectrophotometer (6.6) at a wavelength of 340 nm with air (no cuvet in the beam path) or water as reference. The total volume, V, of test solution in the cuvet shall be 3,02 ml. To calibrate the method, replace the sample e
37、xtract by standard solutions of citric acid monohydrate (5.3) having mass concentrations of 50 mg/l, 25 mg/l and 12,5 mg/l and proceed as described in this clause. Table 1 Pipetting procedure Pipette into cuvets Blank cuvet ml Test cuvet ml Solution 1 according to 5.2.2 Water Sample extract 1,00 2,0
38、0 1,00 1,80 0,20 Mix, read off the absorbance of the solutions (A1) after about 5 min, and start the second reaction by addition of: Solution 2 according to 5.2.3 0,02 0,02 Mix, and on completion of the reaction (after about 5 min), read off the absorbance of the solutions (A2). 8 Calculation In the
39、 reactions on which this determination is based, there is a linear proportionality between the amount of NADH consumed and, consequently, the absorbance difference, A and the concentration by mass of citric acid (see Clause 4). Calculate the absorbance difference using the following equation: 1 2 sa
40、mple 1 2 blank() ()AAA AA= (4) Occasionally a negative value is obtained for the absorbance difference of the blank solution, (A1 A2)blank. In such cases, Equation (4) is still used, and |(A1 A2)blank| is added to (A1 A2)sample. To obtain reliable results, the absorbance difference of the sample ext
41、ract should be at least 0,1. If the absorbance difference of the sample extract is higher than 0,850, the concentration by mass of citric acid in the sample extract is too high. In this case, the sample extract should be diluted until the concentration by mass of citric acid in the cuvet is less tha
42、n 80 g. Calculate the citric acid mass content, C, in grams per litre of the sample extract, using the following equation: CP1000VMFAV= (5) where V is the total volume of test solution in the cuvet, in millilitres (generally 3,02 ml); M is the molar mass of the substance to be determined; F is the d
43、ilution factor of the sample solution; is the absorption coefficient of NADH at 340 nm: 6,30 lmmol1cm1; is the light path of the cuvet, in centimetres; VPis the volume of sample solution used for the preparation of the test solution, in millilitres; BS ISO 20369:2009ISO 20369:2009(E) ISO 2009 All ri
44、ghts reserved 5A is the absorbance difference. If the volumes are the same as in 7.2 and it is unnecessary to dilute the sample extract, calculate the citrate content, C, given as a concentration by mass in grams per litre of sample extract, as anhydrous citric acid (M = 192,1 g/mol) using the follo
45、wing equation: C0,461 A = (6) Calculate the content of anhydrous citric acid as a mass fraction, C, in the wrapping material, as a percentage by mass, using the following equation: CCP100 %= (7) where Pis the mass concentration of the wrapping material sample, in grams per litre of sample extract. I
46、f 1 g of wrapping material is extracted with 100 ml of water, Pis equal to 10 g/l and the content of anhydrous citric acid, C, is given by the following equation: CC10 %= (8) Report the citrate content (mass fraction) as a percentage by mass of anhydrous citric acid in the wrapping material. 9 Preci
47、sion Table 2 shows mean standard deviations of repeatability, sr, and reproducibility, sR, obtained from a collaborative study. The study, involving nine laboratories, was conducted in 2005, using three cigarette paper samples with citrate levels between 0,6 % and 2,5 %. Nine laboratories reported r
48、esults obtained by this method. Table 2 Data analysis of collaborative study Mean value mNsrsRSample n data considered Results expressed as mass fraction in % citric acid monohydrate C1 8 0,68 0,025 0,038 C2 6 2,44 0,046 0,060 C3 8 0,60 0,012 0,022 10 Test report The test report shall include the fo
49、llowing: a) all information required for the complete identification of the sample (type of sample, its origin and its designation); b) reference to this International Standard; c) date of sampling and method; BS ISO 20369:2009ISO 20369:2009(E) 6 ISO 2009 All rights reservedd) details of conditioning; e) date of sample delivery; f) date of analysis; g) moisture content of wrapping material, determined in accordance with ISO 287; h) the analysis r
