BS ISO 18329-2004 Milk and milk products - Determination of furosine content - Ion-pair reverse-phase high-performance liquid chromatography method《乳和乳制品 furosine含量的测定 离子对反相高效液相色谱法.pdf

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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 BS ISO 18329:2004 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 1

2、1 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 participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the r

3、esponsibility 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 publications referred to in this document may be found in the BSI Catalogue under the section entitled “Internatio

4、nal 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 necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British

5、 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 interpretation, or proposals for change, and keep the UK interests informed; monitor related international and E

6、uropean 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 11 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last

7、 issued. Amendments issued since publication Amd. No. Date Comments Reference numbers ISO 18329:2004(E) IDF 193:2004(E) OSI DI dnaF 4002INTERNATIONAL STANDARD ISO 18329 IDF 193 First edition 2004-10-15 Milk and milk products Determination of furosine content Ion-pair reverse- phase high-performance

8、liquid chromatography method Lait et produits laitiers Dtermination de la teneur en furosine Mthode par chromatographie liquide haute performance en phase inverse par paire dions BSISO18329:2004IS:92381 O4002(E) ID:391 F4002(E) DPlcsid Fremia ihTs PDF file may ctnoian emdebt dedyfepcaes. In ccaocnad

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14、uisdehi n Switlrez dnaii ISO dnaID 4002 FA ll rihgtsser edevrBSISO18329:2004IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr iiiContents Page Foreword iv Foreword. v 1 Scope 1 2 Normative references . 1 3 Terms and definitions. 1 4 Principle . 1 5 Reagents 1 6 Apparatus. 2 7

15、Sampling 3 8 Preparation of test portion. 3 8.1 Milk . 3 8.2 Cheese 3 8.3 Hydrolysis 4 8.4 Determination of protein content 4 9 Procedure. 4 9.1 Solid-phase extraction (SPE) of the filtered hydrolysate 4 9.2 HPLC determination 4 10 Calculation and expression of results 6 10.1 Calculation. 6 10.2 Exp

16、ression of results 6 11 Precision 6 11.1 Interlaboratory test . 6 11.2 Repeatability 6 11.3 Reproducibility 6 12 Test report 7 Annex A (informative) Examples of HPLC patterns 8 Annex B (informative) Results of interlaboratory trials 10 Bibliography . 11 BSISO18329:2004IS:92381 O4002(E) ID:391 F002(4

17、)E iv I SO dna ID 4002 F All irhgts seredevrForeword ISO (the International Organization 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 membe

18、r body interested in a subject for which a technical committee has been established 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 Elec

19、trotechnical Commission (IEC) on all matters of electrotechnical standardization. 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 adopt

20、ed by the technical committees are circulated to the member bodies for voting. Publication 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 paten

21、t rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 18329 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

22、. It is being published jointly by ISO and IDF and separately by AOAC International. BSISO18329:2004IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in e

23、very 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 milk products. Draft Internation

24、al 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|IDF 193 was prepared by Techni

25、cal 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 carried out by the Joint ISO/IDF/

26、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). BSISO18329:2004blank BSISO18329:2004NITERNATNOIAL STANDARD IS:92381 O4002(E) ID:391 F002(4)EI

27、 SO dna ID 4002 F All irhgts seredevr 1Milk and 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

28、and milk products. The method is particularly applicable 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 late

29、st edition of the referenced document (including any amendments) applies. ISO 8968-1 IDF 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 sub

30、stance determined by the procedure specified in this 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-hydro

31、lysis into furosine, the determination of which allows 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-R

32、P) HPLC with UV detection at 280 nm. Quantification 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 aci

33、d (HCl), fuming, with a mass fraction 37 %. BSISO18329:2004IS:92381 O4002(E) ID:391 F002(4)E 2 I SO dna ID 4002 F All irhgts seredevr5.2 Hydrochloric acid solution I, c(HCl) = 10,6 mol/l. Mix 8 volumes of hydrochloric acid (5.1) with 1 volume of water to obtain hydrochloric acid solution I. 5.3 Hydr

34、ochloric 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 acid (5.1) with 3 volumes of water to obtain hydrochloric acid

35、solution III. 5.5 Methanol (CH 3 OH). 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 (CH 3 CO 2 H) Dilute 4 ml of glacial acetic acid with water to 1 000 ml. 5.6.3 Potassium chloride solution, c(KCl) = 3 g/l.

36、 Dissolve 3 g of potassium chloride in 1 000 ml of dilute acetic acid (5.6.2). 5.7 Furosine, (e.g. Neosystem 1)or equivalent). 5.8 Furosine standard solution, c -N-(2-furoylmethyl)-L-lysine = 1 nmol/ml (approx.). Dissolve 15 mg of furosine (5.7) in 25 ml of hydrochloric acid solution III (5.4) and m

37、ix. 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 about 1 nmol/ml furosine. Calculate the exact concentratio

38、n 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 Apparatus Usual laboratory equipment and, in particula

39、r, 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. This information is given for the convenience of users

40、of this International Standard and does not constitute an endorsement by ISO or IDF of this product. BSISO18329:2004IS:92381 O4002(E) ID:391 F002(4)E I SO dna ID 4002 F All irhgts seredevr 36.2 HPLC equipment, provided with gradient pumping system, metal-free injector with injection loop between 20

41、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 lower. 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”

42、 column, of diameter 4,6 mm and of length 250 mm, 5 m particle size (e.g. Alltech 2) ), or an equivalent column capable of separating the 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 Analyt

43、ical 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 Pyrex 3)vials, or 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 89

44、68-1 IDF 20-1. 7 Sampling A representative sample should have been sent to the laboratory. It should not have been damaged or changed during 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 Prepara

45、tion 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 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 sol

46、ution 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 constitute an endorsement

47、 by ISO or IDF of these products. BSISO18329:2004IS:92381 O4002(E) ID:391 F002(4)E 4 I SO dna ID 4002 F All irhgts seredevr8.3 Hydrolysis 8.3.1 Bubble the nitrogen (5.9) through the prepared test portion in the screw-cap vial (8.1 or 8.2) for about 2 min. Close the vial tightly and heat its contents

48、 for 23 h in the oven (6.7) set at 110 C. Gently shake the vial after the first hour of heating. 8.3.2 Cool and filter the obtained hydrolysate (8.3.1) through a paper filter (6.9). If necessary, the filtered hydrolysate may be kept for 12 months if stored at 20 C. 8.4 Determination of protein conte

49、nt Determine the nitrogen content in 2 ml of the filtered hydrolysate (8.3.2) by using the “Kjeldahl” method described in ISO 8968-1 IDF 20-1. Calculate the protein content by multiplying the nitrogen content by 6,38. 9 Procedure 9.1 Solid-phase extraction (SPE) of the filtered hydrolysate 9.1.1 Mount the C18 cartridge (6.1) onto the glass syringe (6.10). Pre-wet the cartridge by eluting 5 ml of methanol (5.5) followed

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