1、 Reference numbers ISO 14892:2002(E) IDF 177:2002(E) ISO and IDF 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 vitamine D par chromatog
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6、ng 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 09
7、 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 iiiContents Page Forewordiv, vi Introductionvi 1 Scope 1 2 Normative refere
8、nce1 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 Determination.6 10 Calculation and expression of results.6 10.1 C
9、alculation .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 International Units (IU) 10 Bibliography11 ISO 14892:2002(E) IDF 177:
10、2002(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 bo
11、dy 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 Electrot
12、echnical 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 b
13、y 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 part of ISO 14892 IDF 177 may be the sub
14、ject 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 5, Milk and milk products, and the International Dairy Federation (IDF), in c
15、ollaboration 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 vForeword IDF (the I
16、nternational 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
17、 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 Committees for voting. Publication as an International Standard requires approval by at least 50
18、% 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 International Dairy Federation (IDF), in collaboration with AOAC International. It is being published joi
19、ntly 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. de Vries (NL). ISO 14892:2002(E) IDF 177:2002(E) vi ISO 2002 All rights reserved Introduction
20、 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 the determination of vitamin D in foodstuffs in the Measurements and Testing programme. The study was organiz
21、ed 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 Pharmacopoeia in the monograph on cod-liver oil for the determination of vitamin D in cod-liver oil. Although the Internat
22、ional 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 Standard. Vitamin D is used here as a generic term for vitamin D 2(ergocalciferol) or D 3(cholecalciferol). Th
23、e 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. INTERNATIONAL STANDARD ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved 1Dried skimmed milk Determination of vitam
24、in 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 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
25、 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 , 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
26、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 the internal standard (vitamin D 2 ). 2 Normative reference The following normative document contains provisions which, through
27、 reference in 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
28、 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 of ISO and IEC maintain registers of currently valid International Standards. ISO 3696, Water for analytical laboratory use S
29、pecification 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 skimmed milk mass fraction of substances determined by the procedure specified in this International Standard NOTE It is expres
30、sed 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 separated from impurities by a semi-preparative clean-up using normal-phase HPLC. The vitamin D from the clean-up column is collected
31、. 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 internal standard is added to each test portion prior to saponification. ISO 14892:2002(E) IDF 177:2002(E) 2 ISO 2002 All rights r
32、eserved 5 Reagents 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 (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
33、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) solution. Prepare this solution fresh daily. 5.4 Potassium hydroxide aqueous solution, c(KOH) = 500 g/l. Dissolve 50 g of potassium hydroxi
34、de (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 mix again. Prepare this solution freshly before use. 5.5 Potassium hydroxide aqueous alcoholic solution, c(KOH) = 30 g/l. Dissolve 3 g of potas
35、sium 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 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
36、 (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-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 Antio
37、xidant 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 3standard stock solution, e.g. Ph Eur or USP 1)reference standard cholecalciferol. Accurately weigh ca. 20 mg of cholecalciferol in a 100-ml one
38、-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. 1) Ph Eur and USP are examples of suitable grades of product available commercially. This information is given for the convenience of users
39、 of this International Standard and does not constitute an endorsement by ISO of these products. ISO 14892:2002(E) IDF 177:2002(E) ISO 2002 All rights reserved 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-
40、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 (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
41、 (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. 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
42、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 (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 m
43、ix. 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 IU/ml). 5.18 Referen
44、ce solution Prepare a reference solution by mixing 1,00 ml of the vitamin D 3standard working solution I (5.16.2) and 1,00 ml of the vitamin D 2 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
45、cholecalciferol and 0,2 g/ml of ergocalciferol (8 IU/ml of vitamin D 3and 8 IU/ml of vitamin D 2 ). 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 ana
46、lytical column Use a mixture of acetonitrile/propan-2-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,
47、 capable of weighing to the nearest 0,1 mg, with readability 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 a
48、t the nearest available wavelength for a fixed wavelength 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 i
49、nternal diameter 4,6 mm, packed with 5 m particle size 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
