BS ISO 12080-2-2009 Dried skimmed milk - Determination of vitamin A content - Method using high-performance liquid chromatography《脱脂奶粉 维他命A含量的测定 高效液相色谱法》.pdf

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1、BS ISO12080-2:2009ICS 67.100.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDDried skimmed milk Determination ofvitamin A contentPart 2: Method using high-performanceliquid chromatographyThis British Standardwas published under theauthority of the StandardsPol

2、icy and StrategyCommittee on 31 October2009 BSI 2009ISBN 978 0 580 67130 2Amendments/corrigenda issued since publicationDate CommentsBS ISO 12080-2:2009National forewordThis British Standard is the UK implementation of ISO 12080-2:2009. Itsupersedes BS ISO 12080-2:2000 which is withdrawn.The UK part

3、icipation in its preparation was entrusted to TechnicalCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract.

4、Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 12080-2:2009Reference numbersISO 12080-2:2009(E)IDF 142-2:2009(E)ISO and IDF 2009INTERNATIONAL STANDARD ISO12080-2IDF142-2Second edition2009-10-01Dried skimmed mil

5、k Determination of vitamin A content Part 2: Method using high-performance liquid chromatography Lait crm en poudre Dtermination de la teneur en vitamine A Partie 2: Mthode par chromatographie en phase liquide haute performance BS ISO 12080-2:2009ISO 12080-2:2009(E) IDF 142-2:2009(E) PDF disclaimer

6、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 installed on the computer performing the editing. In downloading this file, parties accept

7、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 the software products used to create this PDF file can be found in the General Info

8、 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 event that a problem relating to it is found, please inform the ISO Central Secretar

9、iat at the address given below. COPYRIGHT PROTECTED DOCUMENT ISO and IDF 2009 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, including photocopying and microfilm, without permission in

10、writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E

11、-mail copyrightiso.org E-mail infofil-idf.org Web www.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2009 All rights reservedBS ISO 12080-2:2009ISO 12080-2:2009(E) IDF 142-2:2009(E) ISO and IDF 2009 All rights reserved iiiForeword ISO (the International Organization for Standard

12、ization) 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 established has the right to

13、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. International Stand

14、ards 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. Publication as an Inte

15、rnational 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 patent rights. ISO 12080-

16、2IDF 142-2 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly by ISO and IDF. ISO 12080IDF 142 consists of the following parts, under the general title Dried skimmed milk

17、 Determination of vitamin A content: Part 1: Colorimetric method Part 2: Method using high-performance liquid chromatography This second edition of ISO 12080-2IDF 142-2 cancels and replaces the first edition (ISO 12080-2:2000), of which it constitutes a minor revision. BS ISO 12080-2:2009ISO 12080-2

18、:2009(E) IDF 142-2:2009(E) iv ISO and IDF 2009 All rights reservedForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having

19、 signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. The main task

20、of Standing Committees is to prepare International Standards. 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 the IDF National Commit

21、tees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 12080-2IDF 142-2 was prepared by the International Dairy Federation (IDF) an

22、d Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by IDF and ISO. All work was carried out by the former Joint ISO-IDF Group of Experts (E46 Vitamins A and D in dried milk) which is now part of the Joint ISO-IDF Action Team on Or

23、ganic contaminants and veterinary residues of the Standing Committee on Analytical methods for additives and contaminants. ISO 12080IDF 142 consists of the following parts, under the general title Dried skimmed milk Determination of vitamin A content: Part 1: Colorimetric method Part 2: Method using

24、 high-performance liquid chromatography This edition of ISO 12080-2IDF 142-2, together with ISO 12080-1IDF 142-1, cancels and replaces IDF 142:1990, of which it constitutes a minor revision. BS ISO 12080-2:2009ISO 12080-2:2009(E) IDF 142-2:2009(E) ISO and IDF 2009 All rights reserved vIntroduction T

25、he methods specified in ISO 12080IDF 142 (all parts) have been selected after consideration and laboratory testing of a variety of alternative procedures. Their advantages include the absence of highly dangerous reagents as in, for example, the Carr-Price method, and the avoidance of reagents that a

26、re not universally available. The decision to provide two separate methods was taken to meet the needs both of laboratories with sophisticated equipment (HPLC) and those without such apparatus. Although the International Standard for vitamin A was discontinued in 1954, the International Unit for thi

27、s substance has continued to be widely used and its use has been maintained in this International Standard. The International Unit for vitamin A was redefined in 1960 as the activity of 0,344 g of pure all-trans-vitamin A acetate (see Annex A). BS ISO 12080-2:2009BS ISO 12080-2:2009INTERNATIONAL STA

28、NDARD ISO 12080-2:2009(E)IDF 142-2:2009(E) ISO and IDF 2009 All rights reserved 1Dried skimmed milk Determination of vitamin A content Part 2: Method using high-performance liquid chromatography WARNING The use of this International Standard may involve hazardous materials, operations and equipment.

29、 Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish health and safety practices and determine the applic

30、ability of regulatory limitations prior to use. 1 Scope This part of ISO 12080IDF 142 specifies a method using high-performance liquid chromatography (HPLC) for the determination of vitamin A in dried skimmed milk containing at least 10 IU (International Units) of vitamin A per gram. 2 Terms and def

31、initions For the purposes of this document, the following terms and definitions apply. 2.1 vitamin A content of dried skimmed milk mass fraction of substances determined by the procedure specified in this part of ISO 12080IDF 142 NOTE Vitamin A content is expressed either in micrograms of retinol pe

32、r gram or in International Units of vitamin A activity per gram. 3 Principle The test sample is saponified and extracted. Vitamin A is separated from impurities by HPLC. The content is determined using an ultraviolet detector or a fluorescence detector. 4 Reagents Use only reagents of recognized ana

33、lytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 4.1 Ethanol (CH3CH2OH), 95 % volume fraction, free from aldehyde. 4.2 Sodium ascorbate solution, 200 g/l. If not available ready-made, prepare by dissolving 3,5 g of ascorbic acid (C6H8O6)

34、in 20 ml of 1 mol/l sodium hydroxide (NaOH) solution and mix. Prepare this solution fresh daily. BS ISO 12080-2:2009ISO 12080-2:2009(E) IDF 142-2:2009(E) 2 ISO and IDF 2009 All rights reserved4.3 Potassium hydroxide aqueous solution (KOH), 50 % mass fraction. Dissolve 50 g of potassium hydroxide in

35、50 ml of water. Mix and cool the solution. Prepare the solution just before use. 4.4 Potassium hydroxide aqueous alcoholic solution, 30 g/l. Dissolve 3 g of potassium hydroxide (KOH) in water and add 10 ml of ethanol (4.1) in a 100 ml one-mark volumetric flask. Make up to the mark with water and mix

36、. Prepare the solution just before use. 4.5 Light petroleum, with a boiling range between 40 C and 60 C, or between 60 C and 80 C. 4.6 Methanol (CH3OH), HPLC grade. 4.7 Mobile phase: mixture of methanol (4.6) and water, 90 + 10 (parts by volume), for example (see Caution in 8.5). 4.8 Vitamin A stand

37、ard solution. Use US Pharmacopeia1)standard reference solution of vitamin A made from crystalline all-trans-retinyl acetate in cottonseed oil, equivalent to 30 mg of retinol (vitamin A alcohol, C20H30O) per gram of oil, or as stated when purchased. Cut the tip from the capsule containing the vitamin

38、 A standard solution and express the oil into a saponification flask. Weigh, to the nearest 0,1 mg, approximately 20 mg of the standard solution. Add 40 ml of ethanol (4.1), 10 ml of sodium ascorbate solution (4.2), and 10 ml of potassium hydroxide aqueous solution (4.3). Saponify and extract as spe

39、cified in 8.3.2 to 8.3.6. Prepare a standard reference solution by proceeding as in 8.4. 4.9 Butylated hydroxytoluene (BHT). 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Liquid chromatograph, fitted with an ultraviolet detector. Typical operating conditions are: vari

40、able UV detector that monitors absorption at 325 nm, or a fixed wavelength detector that monitors at a wavelength of between 300 nm and 360 nm with a detector sensivity of 0,128 AUFS (absorption units, full scale); eluent flow rate of 2 ml/min (at approximately 10 MPa); ambient temperature; injectio

41、n volume of 20 l; chart speed of 10 mm/min. When a fluorescence detector is used, set it at 325 nm for excitation and at 450 nm for emission. 1) Example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO 12080IDF 142 and does not

42、constitute an endorsement by ISO or IDF of this product. BS ISO 12080-2:2009ISO 12080-2:2009(E) IDF 142-2:2009(E) ISO and IDF 2009 All rights reserved 35.2 Chromatogaphic column, of stainless steel, 250 mm 4,6 mm, with 10 m particle size packing of C8 or C18, chemically bonded to totally porous micr

43、osilica particles or a column of equivalent performance. 5.3 Beaker or conical flask, of capacity 250 ml. 5.4 Saponification flask, of capacity approximately 200 ml, fitted with a reflux condenser. 5.5 One-mark volumetric flasks, of capacities 100 ml and 200 ml, ISO 10423class A. 5.6 One-mark pipett

44、es, of capacities 10 ml, 25 ml and 50 ml, ISO 6481class A. 5.7 Steam bath, boiling water bath or electric heating mantle. 5.8 Water bath, capable of operating at a temperature of up to 40 C. 5.9 Separating funnel, of capacity 500 ml, preferably with a polytetrafluoroethylene (PTFE) stopper. 5.10 Ult

45、rasonic bath. 5.11 Filter paper, of diameter 90 mm. 6 Sampling Sampling is not part of the method specified in this part of ISO 12080IDF 142. A recommended sampling method is given in ISO 707IDF 502. A representative sample should have been sent to the laboratory. It should not have been damaged or

46、changed during transport or storage. 7 Preparation of test sample Thoroughly mix the test sample by repeatedly rotating and inverting the sample container. If necessary, transfer the complete test sample to an airtight container of sufficient capacity. 8 Procedure 8.1 General If a check is required

47、of whether the repeatability limit (10.2) is met, carry out two single determinations in accordance with 8.2 to 8.5. For all operations, work in subdued light or use low-actinic glassware. 8.2 Test solution Weigh, to the nearest 0,001 g, about 20 g of dried milk into a beaker or conical flask (5.3)

48、and dissolve in 50 ml of hot water at a temperature of at least 80 C. Break up any lumps with a spatula or by using an ultrasonic bath (5.10). Cool to room temperature. Transfer quantitatively to a 100 ml one-mark volumetric flask (5.5). Make up to the mark with water. BS ISO 12080-2:2009ISO 12080-2

49、:2009(E) IDF 142-2:2009(E) 4 ISO and IDF 2009 All rights reserved8.3 Saponification and extraction 8.3.1 Transfer, by means of a pipette (5.6), 25 ml of the prepared test portion (8.2) to a saponification flask (5.4). Add 20 ml of potassium hydroxide (4.3) and 10 ml of sodium ascorbate solution (4.2). Add 50 ml of ethanol (4.1) and mix well. 8.3.2 Reflux for 30 min on a steam bath (5.7) and swirl from time to time. Cool immediately under running water. 8.3.3 Transfer the li

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