1、Cheese Determination of propionic acid level by chromatography Part 2: Method by ion exchange chromatography Fromages Dtermination de la teneur en acide propionique par chromatographie Partie 2: Mthode par chromatographie par change dions First edition 2017-05 ISO/TS 19046-2 TECHNICAL SPECIFICATION
2、Reference numbers ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) ISO and IDF 2017 IDF/RM 233-2 ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) ii ISO and IDF 2017 All rights reserved COPYRIGHT PROTECTED DOCUMENT ISO and IDF 2017, Published in Switzerland All rights reserved. Unless otherwise specified, no
3、part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member
4、 body in the country of the requester. ISO copyright office International Dairy Federation Ch. de Blandonnet 8 CP 401 Silver Building Bd Auguste Reyers 70/B B-1030 Brussels CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Tel. + 32 2 325 67 40 Fax +41 22 749 09 47 Fax + 32 2 325 67 41 copy
5、rightiso.org infofil-idf.org www.iso.org www.fil-idf.org ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) Forewords .iv 1 Scope . 1 2 Normative references 1 3 T erms and definitions . 1 4 Principle 1 5 Reagents 1 6 Apparatus . 2 7 Sampling 3 8 Preparation of test sample . 3 9 Quantitative determination b
6、y HPLC 3 9.1 Calibration 3 9.2 Sample test . 3 10 Calculation and expression of results . 4 10.1 Calculation of propionic acid in cheese . 4 10.2 Expression of results 4 11 Precision . 4 11.1 Interlaboratory test. 4 11.2 Repeatability . 4 12 Test report . 4 Annex A (informative) Example of HPLC chro
7、matogram 5 Annex B (informative) Interlaboratory test 6 Bibliography 7 ISO and IDF 2017 All rights reserved iii Contents Page ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) Forewords ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO mem
8、ber 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 that committee. International organizations, governmen
9、tal 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. The procedures used to develop this document and those intended for its further maintenance
10、 are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www. iso. org/ directives). Attenti
11、on 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. Details of any patent rights identified during the development of the document will be in the Introduction a
12、nd/or on the ISO list of patent declarations received (see www. iso. org/ patents). Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of ISO specific term
13、s and expressions related to conformity assessment, as well as information about ISOs adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: w ww . i s o. org/ iso/ foreword. html. This document was prepared by Technical Committee I
14、SO/TC 34, Food products, Subcommittee SC 5, Milk and milk products and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. A list of all parts in the ISO/TS 19046 | IDF/RM 233 series can be found on the ISO website. iv ISO and IDF 2017 All rights reserved IDF (the
15、 International Dairy Federation) is a non-profit private sector organization representing the interests of various stakeholders in dairying at the global level. IDF members are organized in National Committees, which are national associations composed of representatives of dairy-related national int
16、erest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and governments/food control authorities. ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis and sampling for milk and milk products. Since 2001, ISO and IDF joi
17、ntly publish their International Standards using the logos and reference numbers of both organizations. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent right
18、s. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www .iso .org/ patents). Any trade name used in this document is information given for the convenience of users and does not const
19、itute and endorsement. ISO/TS 19046-2 | IDF/RM 233-2 was prepared by the IDF Standing Committee on Analytical Methods for Composition and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. The IDF Reviewed method is equal to an ISO Publicly Available Specifi
20、cation (ISO/PAS) or an ISO Technical Specification (ISO/TS) and is therefore published jointly under ISO conditions. The work was carried out by the IDF/ISO Project Group on Propionic acid (C25) of the Standing Committee on Analytical Methods for Composition under the aegis of its project leader P.
21、Trossat (FR). A list of all parts in the ISO/TS 19046 | IDF/RM 233 series can be found on the ISO website. ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) ISO and IDF 2017 All rights reserved v Cheese Determination of propionic acid level by chromatography Part 2: Method by ion exchange chromatography W
22、ARNING This document can involve the use of products and implementation of procedures and equipment of a hazardous nature. This document does not aim to address all the risks related to its use. It is the responsibility of the user of this document to establish appropriate hygiene and safety practic
23、es before using it, and to determine the applicability of any other restrictions. 1 Scope This document specifies a method for the determination of propionic acid level in cheese, using ion exchange chromatography. 2 Normative references There are no normative references in this document. 3 T erms a
24、 nd definiti ons For the purposes of this document, the following terms and definitions apply. ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: availabl
25、e at h t t p :/ www .iso .org/ obp 3.1 level of propionic acid mass fraction of propionic acid determined following the procedure described in this document Note 1 to entry: The level of propionic acid is expressed in mg/100 g of cheese. 4 Principle Aqueous extraction of propionic acid by dilution o
26、f samples in the mobile phase, centrifugation of the solution obtained, and filtration and assay of the propionic acid in the filtrate by ion exchange chromatography. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or wate
27、r of equivalent purity. 5.1 Propionic acid CH 3CH 2 COOH of purity 99,5 % mass fraction. 5.2 Sulfuric acid H 2 SO 4 containing a mass/volume fraction of 98 %. TECHNICAL SPECIFICATION ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) ISO and IDF 2017 All rights reserved 1 the mobile phase (5.4). 6 Apparatu
28、s Usual laboratory equipment and, in particular, the following. 6.1 Grinding or grating device. 6.2 Analytical balance, capable of weighing to the nearest 1 mg, with a readability of 0,1 mg. 6.3 V olumetric flasks, of capacity 50 ml, 100 ml and 5 000 ml. 6.4 Single-use syringes, 1 ml capacity equipp
29、ed with a 0,2 m polyvinylidene fluoride (PVDF) syringe filter. 6.5 Hot plate stirrer. 6.6 Laboratory centrifuge, capable to produce a centrifugal acceleration of approximately 13 000g. 6.7 V acuum filtr ation s y st em, equipped with a 0,2 m filtration membrane. 6.8 HPLC system, with the following e
30、quipment. 6.8.1 Column oven, set at 50 C. 6.8.2 Ion exchange column, Aminex HPX 87 H BIO RAD 1)(column size: 300 mm 7,8 mm, particle size: 9 m) equipped with a Carbo H BIO RAD precolumn or equivalent. 1) Aminex HPX 87 H is an example of a suitable product available commercially. This information is
31、given for the convenience of users of this document and does not constitute an endorsement by ISO or IDF of this product. ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) 5.3 Water, high performance liquid chromatography (HPLC) grade. 5.4 Mobile phase Weigh exactly 10,1 g of sulfuric acid (5.2) and trans
32、fer into a 5 000 ml flask (6.3) already containing 2 l of water (5.3), make up to the mark with water (5.3) and filter using a vacuum filtration system (6.7). 5.5 Standard solutions 5.5.1 Standard stock solution of propionic acid Weigh exactly 200 mg of propionic acid (5.1) in a 100 ml one mark volu
33、metric flask (6.3). Dilute to the mark with the mobile phase (5.4) and filter standard stock solution using a vacuum filtration system (6.7). 5.5.2 Standard solutions of propionic acid Prepare two additional standard solutions by diluting standard stock solution (5.5.1) in mobile phase (5.4). 25 ml
34、of standard stock solution in a 50 ml one mark volumetric flask (6.3). Dilute to the mark with the mobile phase (5.4). 25 ml of standard stock solution in a 100 ml one mark volumetric flask (6.3). Dilute to the mark with 2 ISO and IDF 2017 All rights reserved 6.8.3 Chromatographic conditions Mobile
35、phase: H 2 SO 40,02 mol/l (5.4) kept under helium at 20 ml/min; flow rate: 0,6 ml/min; quantity injected: 10 l; rinse under H 2 SO 40,02 mol/l, inverted column, for 4 h (needed after each analytical series). An example of the HPLC chromatogram profile obtained with these conditions is shown in Annex
36、 A. 6.8.4 UV detector, set at 210 nm. 6.8.5 Injection syringe or injection system, capable to inject 10 l. 6.8.6 Integration system, preferably being computerized. 7 Sampling Sampling is not part of the method specified in this document. A recommended sampling method is given in ISO 707 | IDF 50. It
37、 is important that the laboratory receives a sample that is representative and has not been damaged or changed during transport or storage. 8 Preparation of test sample Prior to analysis, grind or grate the test sample by using an appropriate grinding or grating device (6.1), if necessary, after rem
38、oving the rind, the smear or the mouldy surface layer of the cheese. Weigh, to the nearest 0,1 mg, about 5 g to 10 g of cheese in a glass flask and record the mass m 1 . Add around 35 ml of mobile phase (5.4) to the test sample. Mix with a magnetic stirrer (6.5) for 1 h at 70 C. Cool down the soluti
39、on and transfer into a 50 ml one mark volumetric flask. Dilute to the mark with the mobile phase (5.4). Filter the solution using a 0,2 m syringe filter (6.4). NOTE A centrifugation at 13 000g for 10 min can be applied if difficulties are met with the direct filtration. 9 Quantitative determination
40、by HPLC 9.1 Calibration Inject 10 l of the three standard solutions (5.5) into the HPLC system (6.8) applying chromatographic conditions described in 6.8.3 and calibrate according the manufacturer requirements. 9.2 Sample test Inject 10 l of the test sample prepared as specified in Clause 8 into the
41、 HPLC system (6.8) applying the chromatographic conditions described in 6.8.3. ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) ISO and IDF 2017 All rights reserved 3 (1) where w e is the mass fraction of propionic acid, in mg/100 g, of cheese; P e is the concentration of propionic acid, in g/100 ml, of
42、the cheese-mobile phase mixture calculated by the integrator after calibration; 50 is the final volume of cheese-mobile phase mixture, in ml; m 1 is the mass of the test sample, in g. 10.2 Expression of results The results are expressed in mg/100 g of cheese, without decimals. 11 Precision 11.1 Inte
43、rlaboratory test The values for the repeatability and reproducibility are derived from the results of an interlaboratory test carried out in accordance with ISO 5725-1 and ISO 5725-2 by three laboratories (see Annex B) and therefore shall be considered as indicative only. 11.2 Repeatability An indic
44、ative value of absolute difference between two single test results obtained with the same method on identical test material in the same laboratory by the same operator using the same equipment within a short interval of time of 48 mg of propionic acid/100 g of cheese should be considered. 12 Test re
45、port The test report shall specify: a) all information necessary for the complete identification of the sample; b) the sampling method used, if known; c) the test method used, with reference to this document, i.e. ISO/TS 19046-2 | IDF/RM 233-2; d) all operating details not specified in this document
46、, or regarded as optional, together with details of any incident which might have affected the result(s); e) the test result(s) obtained and, if the repeatability has been checked, the final quoted result obtained. ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) 10 Calculation and expression of results
47、10.1 Calculation of propionic acid in cheese Calculate the mass fraction of propionic acid, w e , expressed in mg/100 g of cheese using Formula (1): 4 ISO and IDF 2017 All rights reserved Annex A (informative) Example of HPLC chromatogram Figure A.1 Example of HPLC chromatogram ISO/TS 19046-2:2017(E
48、) IDF/RM 233-2:2017(E) ISO and IDF 2017 All rights reserved 5 Samples S1 S2 S3 S4 S5 S6 Mean Number of participating laboratories 3 3 3 3 3 3 Mean value (mg propionic acid/100 g of cheese) 391,5 426,8 313,2 522,5 496,2 20,3 Repeatability standard deviation, s r(mg propionic acid/100 g of cheese) 24,
49、1 3,1 19,1 20,2 21,1 0,0 17,4 Repeatability limit, r (2,8 s r ) (mg propionic acid/100 g of cheese) 66,9 8,5 52,8 56,0 58,5 0,0 48,1 ISO/TS 19046-2:2017(E) IDF/RM 233-2:2017(E) Annex B (informative) Interlaboratory test An interlaboratory collaborative test involving three laboratories was carried out on six cheeses samples in blind duplicate. The test samples were prepared and distributed by ACTALIA CECALAIT, Poligny (FR), which also performed the statistical analys
