《ISO-14892-2002.pdf》由会员分享，可在线阅读，更多相关《ISO-14892-2002.pdf（18页珍藏版）》请在三一文库上搜索。
1、 Reference numbers ISO 14892:2002(E) IDF 177:2002(E) ISO and IDF 2002 INTERNATIONAL 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 vitamine D par chromato
2、graphie liquide haute performance ISO 14892:2002(E) IDF 177:2002(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installe
3、d on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobes licensing policy. Neither the ISO Central Secretariat nor the IDF accepts any liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of
4、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 taken to ensure that the file is suitable for use by ISO member bodies and IDF national committees. In the unlikely e
5、vent that a problem relating to it is found, please inform the ISO Central Secretariat at the address given below. ISO and IDF 2002 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, includ
6、ing photocopying and microfilm, without permission in writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 41 Square Vergote B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 0
7、9 47 Fax + 32 2 733 04 13 E-mail copyrightiso.ch E-mail infofil-idf.org Web www.iso.ch Web www.fil- Printed in Switzerland ii ISO and IDF 2002 All rights reserved ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved iii Contents Page Forewordiv, vi Introductionvi 1 Scope1 2 Normative refer
8、ence1 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 portion5 9.3 Saponification and extraction 5 9.4 Purification.6 9.5 Determination.6 10 Calculation and expression of results.6 10.1 Calc
9、ulation .6 10.2 Expression of results7 11 Precision.7 11.1 Interlaboratory test7 11.2 Repeatability 7 11.3 Reproducibility.7 12 Test report7 Annex A (normative) Notes on procedure 8 Annex B (informative) Activity expressed in International Units (IU) 10 Bibliography11 ISO 14892:2002(E) IDF 177:2002(
10、E) iv ISO 2002 All rights reserved Foreword 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 member body in
11、terested 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 Electrotechni
12、cal 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 is to prepare International Standards. Draft International Standards adopted by the
13、 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 part of ISO 14892IDF 177 may be the subject o
14、f patent rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 14892IDF 177 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collabor
15、ation 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. ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved v Foreword IDF (the Interna
16、tional 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 Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the d
17、evelopment 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 Committees for voting. Publication as an International Standard requires approval by at least 50 % of N
18、ational Committees casting a vote. International Standard ISO 14892IDF 177 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
19、 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. de Vries (NL). ISO 14892:2002(E) IDF 177:2002(E) vi ISO 2002 All rights reserved Introduction The me
20、thod 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 the determination of vitamin D in foodstuffs in the Measurements and Testing programme. The study was organized by t
21、he 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 Pharmacopoeia in the monograph on cod-liver oil for the determination of vitamin D in cod-liver oil. Although the International S
22、tandard 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 Standard. Vitamin D is used here as a generic term for vitamin D2 (ergocalciferol) or D3 (cholecalciferol). The Inter
23、national Unit for vitamin D3 is defined as the activity of 0,025 g of pure cholecalciferol, and for vitamin D2 defined as the activity of 0,025 g of ergocalciferol. INTERNATIONAL STANDARD ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved 1 Dried skimmed milk Determination of vitamin D c
24、ontent using high-performance liquid chromatography 1 Scope This International Standard specifies a method for the determination of vitamin D in a test sample containing 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 liqui
25、d chromatography (HPLC). The results of the determination are only reliable if, in the case of determination of vitamin D3, the test sample contains only vitamin D3, and no vitamin D2 (which will be added as an internal standard) and, in the case of determination of vitamin D2, the test sample conta
26、ins only vitamin D2 and no vitamin D3 (which will be added as an internal standard). This is to be verified by the procedure carried out without the addition of the internal standard (vitamin D2). 2 Normative reference The following normative document contains provisions which, through reference in
27、this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibility of applying the
28、most recent edition of the normative document indicated below. For undated references, the latest edition of the normative document referred to applies. Members of ISO and IEC maintain registers of currently valid International Standards. ISO 3696, Water for analytical laboratory use Specification a
29、nd 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 skimmed milk mass fraction of substances determined by the procedure specified in this International Standard NOTE It is expressed either in
30、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 separated from impurities by a semi-preparative clean-up using normal-phase HPLC. The vitamin D from the clean-up column is collected. The content
31、is determined using reverse-phase HPLC with UV detection. Vitamin D2 is used as an internal standard in the determination of vitamin D3 and vice versa. The internal standard is added to each test portion prior to saponification. ISO 14892:2002(E) IDF 177:2002(E) 2 ISO 2002 All rights reserved 5 Reag
32、ents and materials Use only reagents of recognized analytical grade, unless otherwise specified. 5.1 Water, complying with at least grade 1 in accordance with ISO 3696. 5.2 Ethanol (C2H5OH), 95 % (by volume), free from aldehydes. 5.3 Sodium ascorbate solution, c( NaC6H5O6H2O) = 200 g/l. If not avail
33、able ready-made, prepare this solution by dissolving 3,5 g of ascorbic acid (C6H8O6) in 20 ml of 1 mol/l sodium hydroxide (NaOH) 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
34、100 ml volumetric flask (6.6). Mix and cool the obtained solution to ambient temperature. Dilute to the mark with water then mix 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. A
35、dd 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 this 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 (CH3OH), HPLC grade. 5.8 n-Hexa
36、ne (C6H14), HPLC grade. 5.9 n-Pentanol (C5H11OH), HPLC grade. 5.10 Acetonitrile (CH3CN), HPLC grade. 5.11 Propan-2-ol (C3H7OH), 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
37、 n-hexane (5.8). 5.15 Filter paper, of diameter 9 cm. 5.16 Vitamin D3 standard solutions 5.16.1 Vitamin D3 standard stock solution, e.g. Ph Eur or USP1) reference standard cholecalciferol. Accurately weigh ca. 20 mg of cholecalciferol in a 100-ml one-mark volumetric flask (6.6). Dilute to the mark w
38、ith ethanol (5.2) and mix. This solution may be stored in a freezer at a temperature of ( 18 1) C for 1 month. 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 const
39、itute an endorsement by ISO of these products. ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved 3 5.16.2 Vitamin D3 standard working solution I Use a pipette (6.7) to add 10 ml of the vitamin D3 standard stock solution (5.16.1) to a 100-ml one-mark volumetric flask (6.6). Dilute to the
40、 mark with ethanol (5.2) and mix. 5.16.3 Vitamin D3 standard working solution II Use a pipette (6.7) to add 1 ml of the vitamin D3 standard 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 (
41、= 8 IU/ml). 5.17 Vitamin D2 standard solutions 5.17.1 Vitamin D2 standard stock solution, e.g. Ph Eur or USP1) 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 s
42、tored in a freezer for 1 month. 5.17.2 Vitamin D2 standard working solution I Use a pipette (6.7) to add 10 ml of the vitamin D2 standard 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 D2 standard working solution I
43、I Use another pipette (6.7) to add 1 ml of the vitamin D2 standard 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 IU/ml). 5.18 Reference solution Prepare a reference solution by mixing
44、 1,00 ml of the vitamin D3 standard working solution I (5.16.2) and 1,00 ml of the vitamin D2 standard working solution I (5.17.2) in a 100-ml volumetric flask and dilute to the mark with methanol (5.7). This reference solution contains 0,2 g/ml of cholecalciferol and 0,2 g/ml of ergocalciferol (8 I
45、U/ml of vitamin D3 and 8 IU/ml of vitamin D2). 5.19 Mobile phase for clean-up column Degas n-hexane (5.8) under reduced pressure. Mix 3 ml of n-pentanol (5.9) with 997 ml of the just-degassed n-hexane (see 6.3 and A.2.1). 5.20 Mobile phase for analytical column Use a mixture of acetonitrile/propan-2
46、-ol/water (ratio 90:7:3); for example, mix amounts of 900 ml acetonitrile (5.10), 70 ml propan-2-ol (5.11) and 30 ml water (5.1) (see 6.4 and A.3.1). 6 Apparatus Usual laboratory equipment and, in particular, the following. 6.1 Analytical balance, capable of weighing to the nearest 0,1 mg, with read
47、ability to 0,01 mg. 6.2 Liquid chromatograph, fitted with an ultraviolet detector. Typical operating conditions are the following: ISO 14892:2002(E) IDF 177:2002(E) 4 ISO 2002 All rights reserved UV detector that monitors absorption at 265 nm or at the nearest available wavelength for a fixed wavele
48、ngth detector; two columns, clean-up and analytical; detector sensitivity, 0,128 AUFS (absorbance units full scale); chart speed, 10 mm/min; temperature, ambient. 6.3 Clean-up chromatographic column, made of stainless steel, of length 250 mm and internal diameter 4,6 mm, packed with 5 m particle siz
49、e packing of silica (normal-phase material), passing system suitability test (A.2.1); eluent flow rate 3 ml/min; injection volume 600 l. 6.4 Analytical chromatographic column, made of stainless steel, of length 250 mm and internal diameter 4,6 mm, packed with 5 m particle size packing of silica C-18 or ODS (octadecyldimethylsilane) reverse-phase material, passing system suitability test (A.3.1); eluent flow rate 2 ml/min; injection volume 200 l. 6.5 Beakers or conical flasks, of capacity 250 ml.