1、BRITISH STANDARD BS ISO 14892:2002 Dried skimmed milk Determination of vitamin D content using high-performance liquid chromatography ICS 67.100.10 BS ISO 14892:2002 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 2 February 2005 BSI 2 Februa
2、ry 2005 ISBN 0 580 45404 5 National foreword This British Standard reproduces verbatim ISO 14892:2002 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 responsibility to: A list of o
3、rganizations 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 “International Standards Correspondence
4、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 Standard does not of itself
5、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 European developments and prom
6、ulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to vi, pages 1 to 11 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publicati
7、on Amd. No. Date Comments Reference numbers ISO 14892:2002(E) IDF 177:2002(E) OSI DI dnaF 2002INTERNATIONAL STANDARD ISO 14892 IDF 177 First edition 2002-02-01 Dried skimmed milk Determination of vitamin D content using high-performance liquid chromatography Lait crm sec Dtermination de la teneur en
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13、Tle. + 3289 337 2 88 Fxa 90 947 22 14 + 74 Fxa 40 337 2 23 + 31 E-mali copyrightiso.ch -Emalini fof-lidif.org We bwww.is.och We bwww.fil-.grocom rPinetdni wS ztierland ii ISO dna ID 2002 FA ll irghtsser e devrBSISO14892:2002IS:29841 O2002(E) ID:771 F2002(E) I SO 2002 All irthgs ersedevr iiiContents
14、Page Forewordiv, v Introductionvi 1 Scope 1 2 Normative reference1 3 Term and definition .1 4 Principle1 5 Reagents and materials 2 6 Apparatus .3 7 Sampling.4 8 Preparation of test sample4 9 Procedure .5 9.1 General5 9.2 Test portion 5 9.3 Saponification and extraction 5 9.4 Purification .6 9.5 Det
15、ermination.6 10 Calculation and expression of results.6 10.1 Calculation .6 10.2 Expression of results 7 11 Precision.7 11.1 Interlaboratory test7 11.2 Repeatability 7 11.3 Reproducibility.7 12 Test report 7 Annex A (normative) Notes on procedure 8 Annex B (informative) Activity expressed in Interna
16、tional Units (IU) 10 Bibliography11 BSISO14892:2002IS:29841 O2002(E) ID:771 F2002(E) iv I SO 2002 All irthgs ersedevr Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Stan
17、dards 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, governmental and non-governmental, in liaison with ISO, also t
18、ake part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 3. The main task of technical committees
19、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 requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibil
20、ity that some of the elements of this part of ISO 14892 IDF 177 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 14892 IDF 177 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC
21、 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. Annex A forms a normative part of this International Standard. Annex B is for information only. BSISO
22、14892:2002IS:29841 O2002(E) ID:771 F2002(E) I SO 2002 All irthgs ersedevr vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committee has the right to be represented on the IDF Standing C
23、ommittees 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 International Standards adopted by the Action Teams and Standing Committees are circulated to the National Com
24、mittees for voting. Publication as an International Standard requires approval by at least 50 % of National Committees casting a vote. International Standard ISO 14892 IDF 177 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the Internation
25、al 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/AOAC Action Team, Selected food additives and vitamins, under the aegis of its project leader, Mr E.J.
26、 de Vries (NL). BSISO14892:2002IS:29841 O2002(E) ID:771 F2002(E) vi I SO 2002 All irthgs ersedevr Introduction The method described in this International Standard was selected after consideration and laboratory testing of a variety of alternative procedures in an interlaboratory study of methods for
27、 the determination of vitamin D in foodstuffs in the Measurements and Testing programme. The study was organized by the Community Bureau of Reference (BCR) of the Commission of the European Communities. The method is based upon the same principles as the method described in the European Pharmacopoei
28、a in the monograph on cod-liver oil for the determination of vitamin D in cod-liver oil. Although the International Standard for vitamin D was withdrawn in 1984, the International Unit for this substance has continued to be widely used and therefore its use has been maintained in this International
29、Standard. Vitamin D is used here as a generic term for vitamin D 2(ergocalciferol) or D 3(cholecalciferol). The International Unit for vitamin D 3is defined as the activity of 0,025 g of pure cholecalciferol, and for vitamin D 2defined as the activity of 0,025 g of ergocalciferol. BSISO14892:2002INT
30、ENRATIONAL TSANDADR IS:29841 O2002(E) ID:771 F2002(E)I SO 2002 All irthgs ersedevr 1Dried skimmed milk Determination of vitamin D content using high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination of vitamin D in a test sample containing
31、 at least 10 g of vitamin D per 100 g equal to 400 International Units (IU) of vitamin D per 100 g by using high- performance liquid chromatography (HPLC). The results of the determination are only reliable if, in the case of determination of vitamin D 3 , the test sample contains only vitamin D 3 ,
32、 and no vitamin D 2(which will be added as an internal standard) and, in the case of determination of vitamin D 2 , the test sample contains only vitamin D 2and no vitamin D 3(which will be added as an internal standard). This is to be verified by the procedure carried out without the addition of th
33、e internal standard (vitamin D 2 ). 2 Normative reference The following normative document contains provisions which, through reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do no
34、t apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Members
35、of ISO and IEC maintain registers of currently valid International Standards. ISO 3696, Water for analytical laboratory use Specification and test methods 3 Term and definition For the purposes of this International Standard, the following term and definition apply. 3.1 vitamin D content of dried sk
36、immed milk mass fraction of substances determined by the procedure specified in this International Standard NOTE It is expressed either in micrograms per gram or in International Units (IU) of vitamin D activity per gram. 4 Principle The test sample is saponified and extracted. Vitamin D is separate
37、d from impurities by a semi-preparative clean-up using normal-phase HPLC. The vitamin D from the clean-up column is collected. The content is determined using reverse-phase HPLC with UV detection. Vitamin D 2is used as an internal standard in the determination of vitamin D 3and vice versa. The inter
38、nal standard is added to each test portion prior to saponification. BSISO14892:2002IS:29841 O2002(E) ID:771 F2002(E) 2 I SO 2002 All irthgs ersedevr 5 Reagents and materials Use only reagents of recognized analytical grade, unless otherwise specified. 5.1 Water, complying with at least grade 1 in ac
39、cordance with ISO 3696. 5.2 Ethanol (C 2 H 5 OH), 95 % (by volume), free from aldehydes. 5.3 Sodium ascorbate solution, c( NaC 6 H 5 O 6 H 2 O) = 200 g/l. If not available ready-made, prepare this solution by dissolving 3,5 g of ascorbic acid (C 6 H 8 O 6 ) in 20 ml of 1 mol/l sodium hydroxide (NaOH
40、) solution. Prepare this solution fresh daily. 5.4 Potassium hydroxide aqueous solution, c(KOH) = 500 g/l. Dissolve 50 g of potassium hydroxide (KOH) in 50 ml of water in a 100 ml volumetric flask (6.6). Mix and cool the obtained solution to ambient temperature. Dilute to the mark with water then mi
41、x again. Prepare this solution freshly before use. 5.5 Potassium hydroxide aqueous alcoholic solution, c(KOH) = 30 g/l. Dissolve 3 g of potassium hydroxide (KOH) in water. Add 10 ml of ethanol (5.2) in a 100 ml one-mark volumetric flask (6.6). Dilute with water to the 100 ml mark and mix. Prepare th
42、is solution freshly before use. 5.6 Light petroleum, with a boiling range of between 40 C and 60 C, or of between 60 C and 80 C. 5.7 Methanol (CH 3 OH), HPLC grade. 5.8 n -Hexane (C 6 H 14 ), HPLC grade. 5.9 n -Pentanol (C 5 H 11 OH), HPLC grade. 5.10 Acetonitrile (CH 3 CN), HPLC grade. 5.11 Propan-
43、2-ol (C 3 H 7 OH), HPLC grade. 5.12 Nitrogen, chemically pure (oxygen free). 5.13 Butylated hydroxytoluene (BHT), chemically pure. 5.14 Antioxidant solution, 10 mg BHT (5.13) per milliltre of n-hexane (5.8). 5.15 Filter paper, of diameter 9 cm. 5.16 Vitamin D 3standard solutions 5.16.1 Vitamin D 3st
44、andard stock solution, e.g. Ph Eur or USP 1)reference standard cholecalciferol. Accurately weigh ca. 20 mg of cholecalciferol in a 100-ml one-mark volumetric flask (6.6). Dilute to the mark with ethanol (5.2) and mix. This solution may be stored in a freezer at a temperature of ( 18 1) C for 1 month
45、. 1) Ph Eur and USP are examples of suitable grades of product available commercially. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products. BSISO14892:2002IS:29841 O2002(E) ID:771 F2002(E) I SO 2002 All
46、 irthgs ersedevr 35.16.2 Vitamin D 3standard working solution I Use a pipette (6.7) to add 10 ml of the vitamin D 3standard stock solution (5.16.1) to a 100-ml one-mark volumetric flask (6.6). Dilute to the mark with ethanol (5.2) and mix. 5.16.3 Vitamin D 3standard working solution II Use a pipette
47、 (6.7) to add 1 ml of the vitamin D 3standard working solution I (5.16.2) to a second 100-ml one-mark volumetric flask (6.6). Dilute to the mark with ethanol (5.2) and mix. This solution contains 0,2 g/ml (= 8 IU/ml). 5.17 Vitamin D 2standard solutions 5.17.1 Vitamin D 2standard stock solution, e.g.
48、 Ph Eur or USP 1 )reference standard ergocalciferol. Accurately, weigh ca. 20 mg of ergocalciferol in a 100-ml one-mark volumetric flask. Dilute to the mark with ethanol (5.2) and mix. This solution may be stored in a freezer for 1 month. 5.17.2 Vitamin D 2standard working solution I Use a pipette (
49、6.7) to add 10 ml of the vitamin D 2standard stock solution (5.17.1) to a 100-ml one-mark volumetric flask (6.6). Dilute to the mark with ethanol (5.2) and mix. 15.17.3 Vitamin D 2standard working solution II Use another pipette (6.7) to add 1 ml of the vitamin D 2standard working solution I (5.17.2) to a second 100-ml one- mark volumetric flask (6.6). Dilute to the mark with ethanol (5.2) and mix. This solution contains 0,2 g/ml (8
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