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1、BRITISH STANDARD BS ISO 18329:2004 Milk and milk products Determination of furosine content Ion-pair reverse-phase high-performance liquid chromatography method ICS 67.100.01 ? Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI BS ISO 18329:200
2、4 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 11 November 2004 BSI 11 November 2004 ISBN 0 580 44743 X National foreword This British Standard reproduces verbatim ISO 18329:2004 and implements it as the UK national standard. The UK partic
3、ipation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international publi
4、cations referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the nec
5、essary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible international/European committee any enquiries on the int
6、erpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to
7、11 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. DateComments Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference numbers
8、 ISO 18329:2004(E) IDF 193:2004(E) OSI DI dnaF 4002 INTERNATIONAL STANDARD ISO 18329 IDF 193 First edition 2004-10-15 Milk and milk products Determination of furosine content Ion-pair reverse- phase high-performance liquid chromatography method Lait et produits laitiers Dtermination de la teneur en
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15、We bwww.fil-idf.o gr Plbuisdehi n Switlrez dna ii ISO dnaID 4002 FA ll rihgtsser edevr BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr iii Contents Pa
16、ge Forewordiv Foreword.v 1 Scope1 2 Normative references .1 3 Terms and definitions.1 4 Principle.1 5 Reagents1 6 Apparatus.2 7 Sampling3 8 Preparation of test portion.3 8.1 Milk .3 8.2 Cheese3 8.3 Hydrolysis4 8.4 Determination of protein content 4 9 Procedure.4 9.1 Solid-phase extraction (SPE) of t
17、he filtered hydrolysate4 9.2 HPLC determination4 10 Calculation and expression of results6 10.1 Calculation.6 10.2 Expression of results6 11 Precision6 11.1 Interlaboratory test .6 11.2 Repeatability6 11.3 Reproducibility6 12 Test report7 Annex A (informative) Examples of HPLC patterns8 Annex B (inf
18、ormative) Results of interlaboratory trials10 Bibliography .11 BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:92381 O4002(E) ID:391 F002(4)E iv I SO dna ID 4002 F All irhgts seredevr Foreword ISO (the International Orga
19、nization for Standardization) is a worldwide 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 establis
20、hed has the right to be represented on that 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
21、. International Standards are drafted in accordance 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. P
22、ublication as an International Standard requires 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 pate
23、nt rights. ISO 18329IDF 193 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC Internation
24、al. BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr v Foreword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector
25、with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and mi
26、lk products. Draft International Standards adopted by the Action Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50% of IDF National Committees casting a vote. International Standard ISO 18329|
27、IDF 193 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. All work was car
28、ried out by the Joint ISO/IDF/AOAC Action Team, Characterization of heat treatment, of the Standing Committee on Minor components and characterization of physical properties, under the aegis of its project leader, Prof. P. Resmini (IT). BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, M
29、on Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI blank BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI NITERNATNOIAL STANDARD IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr 1 Milk and
30、milk products Determination of furosine content Ion-pair reverse-phase high-performance liquid chromatography method 1 Scope This International Standard specifies a method for the quantitative determination of furosine (-furoylmethyl- lysine) in milk and milk products. The method is particularly app
31、licable to raw or heat-treated milk and to cheese. 2 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
32、any amendments) applies. ISO 8968-1IDF 20-1, Milk Determination of nitrogen content Part 1 Kjeldahl method 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 furosine content mass fraction of substance determined by the procedure specified in th
33、is International Standard NOTE Furosine content is expressed in milligrams per 100 g of protein. 4 Principle The first stable “Maillard Reaction” (MR) product formed in milk and in cheese, -lactulosyl-lysine, is partially converted by warm acid-hydrolysis into furosine, the determination of which al
34、lows the extent of the early stage of MR to be evaluated. The MR extent is related to the type and intensity of heat treatments applied both to raw material and in processing. The determination of furosine is performed by ion-pair reverse-phase (IP-RP) HPLC with UV detection at 280 nm. Quantificatio
35、n of furosine is obtained by reference to a standard sample of furosine. 5 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 5.1 Hydrochloric acid (HCl), fuming, with a mass fraction 37 %. BS ISO
36、 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:92381 O4002(E) ID:391 F002(4)E 2 I SO dna ID 4002 F All irhgts seredevr 5.2 Hydrochloric acid solution I, c(HCl) = 10,6 mol/l. Mix 8 volumes of hydrochloric acid (5.1) with 1 vol
37、ume of water to obtain hydrochloric acid solution I. 5.3 Hydrochloric acid solution II, c(HCl) = 8,0 mol/l. Mix 2 volumes of hydrochloric acid (5.1) with 1 volume of water to obtain hydrochloric acid solution II. 5.4 Hydrochloric acid solution III, c(HCl) = 3,0 mol/l. Mix 1 volume of hydrochloric ac
38、id (5.1) with 3 volumes of water to obtain hydrochloric acid solution III. 5.5 Methanol (CH3OH). 5.6 HPLC elution solvents Prepare HPLC elution solvents by using HPLC-grade reagents. 5.6.1 Water, of HPLC-grade. 5.6.2 Dilute acetic acid (CH3CO2H) Dilute 4 ml of glacial acetic acid with water to 1 000
39、 ml. 5.6.3 Potassium chloride solution, c(KCl) = 3 g/l. Dissolve 3 g of potassium chloride in 1 000 ml of dilute acetic acid (5.6.2). 5.7 Furosine, (e.g. Neosystem1) or equivalent). 5.8 Furosine standard solution, c-N-(2-furoylmethyl)-L-lysine = 1 nmol/ml (approx.). Dissolve 15 mg of furosine (5.7)
40、in 25 ml of hydrochloric acid solution III (5.4) and mix. Dilute 5 ml of this solution with the hydrochloric acid solution III (5.4) to 100 ml. Mix again to obtain dilution 1. Dilute 1 ml of dilution 1 with hydrochloric acid solution III (5.4) to 100 ml to obtain a furosine standard solution of abou
41、t 1 nmol/ml furosine. Calculate the exact concentration of furosine in the final furosine standard solution on the basis of the net content declared for the commercial product. The furosine standard solution remains stable for 24 months, if stored at 20 C. 5.9 Nitrogen, gas chromatography purity. 6
42、Apparatus Usual laboratory equipment and, in particular, the following. 6.1 C 18 cartridge, or minicolumn (500 mg), used in the solid-phase extraction. 1) Furosine is supplied by Neosystem, Rue de Bologne 7, 67100 Strasbourg, France. This is an example of a suitable product available commercially. T
43、his information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO or IDF of this product. BS ISO 18329:2004 Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:40:43 GMT+00:00 2006, Uncontrolled Copy, (c) BSI IS:92381 O4002(E) ID
44、:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr 3 6.2 HPLC equipment, provided with gradient pumping system, metal-free injector with injection loop between 20 l and 50 l, and a thermostatic column oven. 6.3 UV-detector, capable of operating at 280 nm wavelength with minimum AUFS of 0,010 or lo
45、wer. Under these chromatographic conditions and with an injection of 10 pmol of furosine, the signal-to-noise ratio shall be higher than 10. 6.4 “Furosine dedicated” column, of diameter 4,6 mm and of length 250 mm, 5 m particle size (e.g. Alltech2), or an equivalent column capable of separating the
46、furosine peak on the baseline without interfering with other peaks. 6.5 Integrator, or data-reprocessing software, capable of measuring the peak areas. 6.6 Analytical balance, capable of weighing to the nearest 1 mg. 6.7 Oven, capable of being maintained at 110 C 2 C. 6.8 Screw-cap Pyrex3) vials, or
47、 other heat-resistant sealing vials. 6.9 Paper filters, of medium porosity. 6.10 Glass syringe, of capacity of 10 ml. 6.11 Kjeldahl apparatus,conforming to ISO 8968-1IDF 20-1. 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed durin
48、g transport or storage. Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707. 8 Preparation of test portion 8.1 Milk Pipette 2 ml of test sample into a screw-cap Pyrex-vial (6.8). Add 6 ml of hydrochloric solution I (5.2) and
49、mix. 8.2 Cheese Weigh an aliquot of test sample corresponding to an amount of 40 mg to 50 mg of protein into a screw-cap vial (6.8). Add 8 ml of hydrochloric solution II (5.3) and mix. 2) The “Furosine dedicated” column is supplied by Alltech-Europe, Laarne, Belgium. 3) Pyrex is an example of a suitable product available commercially. This information is given for the convenience of users of this International Standard and does not constitut