1、BS ISO12080-1:2009ICS 67.100.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDDried skimmed milk Determination ofvitamin A contentPart 1: Colorimetric methodThis British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 31
2、 October2009 BSI 2009ISBN 978 0 580 67129 6Amendments/corrigenda issued since publicationDate CommentsBS ISO 12080-1:2009National forewordThis British Standard is the UK implementation of ISO 12080-1:2009. Itsupersedes BS ISO 12080-1:2000 which is withdrawn.The UK participation in its preparation wa
3、s 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. Users are responsible for its c
4、orrect application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 12080-1:2009Reference numbersISO 12080-1:2009(E)IDF 142-1:2009(E)ISO and IDF 2009INTERNATIONAL STANDARD ISO12080-1IDF142-1Second edition2009-10-01Dried skimmed milk Determination of vitamin A co
5、ntent Part 1: Colorimetric method Lait crm en poudre Dtermination de la teneur en vitamine A Partie 1: Mthode colorimtrique BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobes licensing policy, this file may
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9、s 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 writing from either ISO or IDF at the respective address below. ISO copyright office International Dairy
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11、nd ii ISO and IDF 2009 All rights reservedBS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) ISO and IDF 2009 All rights reserved iiiForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of prepari
12、ng 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 be represented on that committee. International organizations, governmental and non-governmental, in lia
13、ison with ISO, also take 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 2. The main task of
14、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 International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is
15、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 12080-1IDF 142-1 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and mil
16、k 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 Determination of vitamin A content: Part 1: Colorimetric method Part 2: Method using high-performance l
17、iquid chromatography This second edition of ISO 12080-1IDF 142-1 cancels and replaces the first edition (ISO 12080-1:2000), of which it constitutes a minor revision. BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) iv ISO and IDF 2009 All rights reservedForeword IDF (the International Dairy
18、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 signed a formal agreement on cooperation with IDF. All members of IDF have the right to be represented
19、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 of Standing Committees is to prepare International Standards. Draft International Standards adopted by t
20、he 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 Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document ma
21、y be the subject of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 12080-1IDF 142-1 was prepared by the International Dairy Federation (IDF) and Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being p
22、ublished 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 Organic contaminants and veterinary residues of the Standing Committee on Analytical methods for additives
23、 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 high-performance liquid chromatography This edition of ISO 12080-1IDF 142-1, together with ISO 12080-2I
24、DF 142-2, cancels and replaces IDF 142:1990, of which it constitutes a minor revision. BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) ISO and IDF 2009 All rights reserved vIntroduction The methods specified in ISO 12080IDF 142 (all parts) have been selected after consideration and laborato
25、ry 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 are not universally available. The decision to provide two separate methods was taken to meet the needs b
26、oth 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 this substance has continued to be widely used and its use has been maintained in this International Standa
27、rd. 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 12080-1:2009BS ISO 12080-1:2009INTERNATIONAL STANDARD ISO 12080-1:2009(E)IDF 142-1:2009(E) ISO and IDF 2009 All rights reserved 1Dried skimmed milk Dete
28、rmination of vitamin A content Part 1: Colorimetric method WARNING The use of this International Standard may involve hazardous materials, operations and equipment. Persons using this International Standard should be familiar with normal laboratory practice. This standard does not purport to address
29、 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 applicability of regulatory limitations prior to use. 1 Scope This part of ISO 12080IDF 142 specifies a colorimetric method for the determinat
30、ion of vitamin A in dried skimmed milk containing at least 10 IU (International Units) of vitamin A per gram. 2 Terms and definitions 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 th
31、e procedure specified in this part of ISO 12080IDF 142 NOTE Vitamin A content is expressed either in micrograms of retinol per gram or in International Units of vitamin A activity per gram. 3 Principle The test sample is saponified and extracted. The unsaponifiable matter is reacted with trifluoroac
32、etic acid. The absorbance at 620 nm is measured. 4 Reagents Use only reagents of recognized analytical 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 solutio
33、n, 200 g/l. If not available ready-made, prepare by dissolving 3,5 g of ascorbic acid (C6H8O6) in 20 ml of 1 mol/l sodium hydroxide (NaOH) solution and mix. Prepare this solution fresh daily. BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) 2 ISO and IDF 2009 All rights reserved4.3 Potassium
34、 hydroxide aqueous solution (KOH), 50 % mass fraction. Dissolve 50 g of potassium hydroxide in 50 ml of water. Mix and cool the solution. Prepare this 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 m
35、l of ethanol (4.1) in a 100 ml one-mark volumetric flask. Make up to the mark with water and mix. Prepare this 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 Chloroform (CHCl3). 4.7 Trifluoroacetic acid (CF3COOH). WARNING Chlo
36、roform and trifluoroacetic acid are carcinogenic. Take all necessary precautions. 4.8 Colour reagent. Mix one volume of pure trifluoroacetic acid (4.7) with two volumes of chloroform (4.6). 4.9 Vitamin A standard solution. Use US Pharmacopeia1)standard reference solution of vitamin A made from cryst
37、alline 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. A secondary standard solution may be used if standardized against this primary standard reference solution or by UV measurement. Cut the tip f
38、rom the capsule containing the vitamin A standard reference solution and express the oil into a (tared) 100 ml amber-coloured one-mark volumetric flask. Weigh the contents to the nearest 0,1 mg. Make up to the mark with chloroform (4.6). Use the vitamin A standard solution as soon as possible. Disca
39、rd the solution after 8 h. 4.10 Butylated hydroxytoluene (BHT). 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Photoelectric colorimeter or spectrometer, with an optical mechanism or filter for a wavelength of 620 nm (vitamin A derivative). Use matched absorption cells
40、. An instrument providing linearity between absorbance and concentration is to be preferred. 5.2 Beaker or conical flask, of capacity 250 ml. 5.3 Saponification flask, of capacity approximately 200 ml, fitted with a reflux condenser. 5.4 One-mark volumetric flasks, of capacities 100 ml and 200 ml, I
41、SO 10423class A. 5.5 One-mark pipettes, of capacities 2 ml, 10 ml, 25 ml and 50 ml, ISO 6481class A. 5.6 Automatic pipettes, suitable for organic solvents, ISO 8655-26, or a pipette, to deliver 10 ml. 1) Example of a suitable product available commercially. This information is given for the convenie
42、nce of users of this part of ISO 12080IDF 142 and does not constitute an endorsement by ISO or IDF of this product. BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) ISO and IDF 2009 All rights reserved 35.7 Steam bath, boiling water bath or electric heating mantle. 5.8 Water bath, capable of
43、 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 Ultrasonic 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 reco
44、mmended 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 changed during transport or storage. 7 Preparation of test sample Thoroughly mix the test sample by repeatedly rotating and inverting the sample con
45、tainer. If necessary, transfer the complete test sample to an airtight container of sufficient capacity. 8 Procedure 8.1 General If a check is required 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 sub
46、dued light or use low-actinic glassware. 8.2 Test solution Weigh, to the nearest 0,001 g, about 20 g of the test sample into a beaker or conical flask (5.2) and dissolve in 50 ml of water at a temperature of least 80 C. Break up any lumps with a spatula or by using an ultrasonic bath (5.10). Cool to
47、 room temperature. Transfer quantitatively to a 100 ml one-mark volumetric flask (5.4). Make up to the mark with water. 8.3 Saponification and extraction 8.3.1 Transfer, by means of a pipette (5.5), 25 ml of the prepared test solution (8.2) to a saponification flask (5.3). Add 20 ml of potassium hyd
48、roxide solution (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), swirling from time to time. Cool immediately under running water. 8.3.3 Transfer the liquid to a separating funnel (5.9), and wash twice, each t
49、ime using 30 ml of water, 10 ml of ethanol (4.1), and 40 ml of light petroleum (4.5). Shake vigorously for 30 s and allow to stand until the two layers are clear. BS ISO 12080-1:2009ISO 12080-1:2009(E) IDF 142-1:2009(E) 4 ISO and IDF 2009 All rights reservedTransfer the aqueous (lower) phase to a second separating funnel and shake with a mixture of 10 ml of ethanol (4.1) and 40 ml of light petroleum (4.5). Leave to separate. 8.3.4 Transfer the aqueous phase to a third separating funne
