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1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS ISO 12080-2:2000 ICS 6
2、7.100.10 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Dried skimmed milk Determination of vitamin A content Part 2: Method using high-performance liquid chromatography Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI
3、 This British Standard, having been prepared under the direction of the Consumer Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 July 2000 BSI 07-2000 ISBN 0 580 36178 0 BS ISO 12080-2:2000 Amendments issued since publi
4、cation Amd. No.DateComments National foreword This British Standard reproduces verbatim ISO 12080-2:2000 and implements it as the UK national standard. The UK participation in its preparation was entrusted to Technical Committee AW/5, Chemical analysis of milk and milk products, which has the respon
5、sibility to: 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 promulgate them in the UK. A li
6、st of organizations 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 Standards Catalogue under the section entitled International Standards
7、 Correspondence Index, or by using the Find facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does
8、 not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the ISO title page, pages ii to iv, pages 1 to 7 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Li
9、censed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI Reference number ISO 12080-2:2000(E) INTERNATIONAL STANDARD ISO 12080-2 First edition 2000-06-01 Dried skimmed milk Determination of vitamin A content Part 2: Method using high-performance liquid
10、 chromatography Lait crm en poudre Dtermination de la teneur en vitamine A Partie 2: Mthode par chromatographie en phase liquide haute performance Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) ii? Licensed Copy: sheffie
11、ldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) ?iii 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
12、 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 take part
13、 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. Draft International Standards adopted by the t
14、echnical 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 12080 may be the subject of patent
15、rights. ISO shall not be held responsible for identifying any or all such patent rights. International Standard ISO 12080-2 was prepared by Technical Committee ISO/TC 34,Agricultural food products, Subcommittee SC 5,Milk and milk products,in collaboration with the International Dairy Federation (IDF
16、) and AOAC International, and will also be published by these organizations. ISO 12080 consists of the following parts, under the general titleDried skimmed milk Determination of vitamin A content: ?Part 1: Colorimetric method ?Part 2: Method using high-performance liquid chromatography Annex A of t
17、his part of ISO 12080 is for information only. Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) iv? Introduction The methods specified in ISO 12080 have been selected after consideration and laboratory testing of a variety
18、 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 both of laboratories wit
19、h 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 Standard. The International U
20、nit for vitamin A was redefined in 1960 as the activity of 0,000 344 mg of pure all-trans-vitamin A acetate (see annex A). Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI INTERNATIONAL STANDARD?ISO 12080-2:2000(E) ?1 Dried skimmed milk Deter
21、mination 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. This standard does not purport to address all the safety problems associated with its use. It is the res
22、ponsibility of the user of this standard to establish safety and health practices and determine the applicability of regulatory limitations prior to use. 1?Scope This part of ISO 12080 specifies a method using high-performance liquid chromatography (HPLC) for the determination of vitamin A in dried
23、skimmed milk containing at least 10 IU (International Units) of vitamin A per gram. 2?Term and definition For the purposes of this part of ISO 12080, the following term and definition apply. 2.1 vitamin A content of dried skimmed milk mass fraction of substances determined by the procedure specified
24、 in this part of ISO 12080 NOTE?It is expressed either in micrograms of retinol per gram or in International Units of vitamin A activity per gram. 3Principle The test sample is saponified and extracted. Vitamin A is separated from impurities by HPLC. The content is determined using an ultraviolet de
25、tector or a fluorescence detector. 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 % (by volume), free from aldehyde. 4.2?Sodium ascorbate solution, 200 g/l. If not
26、 available ready-made, prepare this 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. 4.3?Potassium hydroxide aqueous solution, 50 % (by mass). Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46
27、 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) 2? Dissolve 50 g of potassium hydroxide (KOH) in 50 ml of water. Mix and cool the obtained solution. Prepare the solution freshly before use. 4.4?Potassium hydroxide aqueous alcoholic solution, 30 g/l. Dissolve 3 g of potassium hydroxid
28、e (KOH) in water and add 10 ml of ethanol (4.1) in a 100 ml one-mark volumetric flask. Dilute with water to the 100 ml mark and mix. Prepare the solution freshly before use. 4.5?Light petroleum, with a boiling range of between 40 C and 60 C, or of between 60 C and 80 C. 4.6?Methanol (CH3OH), HPLC gr
29、ade. 4.7?Mobile phase: mixture of methanol and water, ratio 90:10 (by volume), for example (see note in 8.5). 4.8?Vitamin A standard solution Use US Pharmacopeia standard1)reference solution of vitamin A made from crystalline all-trans-retinyl acetate in cottonseed oil, equivalent to 30 mg of retino
30、l (vitamin A alcohol, C20H30O) per gram of oil, or as stated when purchased. Cut the tip from the capsule containing the vitamin 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)
31、, 10 ml of sodium ascorbate solution (4.2) and 10 ml of potassium hydroxide solution (4.3). Saponify and extract as described 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
32、, the following. 5.1?Liquid chromatograph, fitted with an ultraviolet detector. Typical operating conditions are: ?variable 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 AUF
33、S (absorption units, full scale); ?eluent flow rate of 2 ml/min (at approximately 100 atm); ?ambient temperature; ?injection 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)The reference vitamin A solutio
34、n from United States Pharmacopeia Convention, Inc., 12601 Twinbrook Parkway, Rockville, Maryland 20852, USA, is an example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO 12080 and does not constitute an endorsement by ISO of t
35、his product. Licensed Copy: sheffieldun sheffieldun, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) ?3 5.2?Chromatogaphic column, made of stainless steel, 250 mm ? 4,6 mm, packed with 10 ?m particle size packing of C8 or C18, chemically bonded to totally porou
36、s microsilica 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. 5.6?One-mark pipettes, of capac
37、ities 10 ml, 25 ml and 50 ml. 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 polytetrafluorethylene (PTFE) stopper. 5.10Ultrasonic bath 5.11Filter paper, o
38、f diameter 9 cm. 6?Sampling Sampling is not part of the method specified in this International Standard. A recommended sampling method is given in ISO 707 1. It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or sto
39、rage. 7?Preparation of test sample Thoroughly mix the test sample by repeatedly rotating and inverting the sample container. If necessary for this, transfer the complete test sample to an airtight container of sufficient capacity. 8?Procedure 8.1? General NOTE?If it is required to check whether the
40、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) and dissolve i
41、n 50 ml of hot water at a temperature of at least 80 C. Break down 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). Dilute with water to the 100 ml mark. Licensed Copy: sheffieldun sheffield
42、un, na, Mon Nov 27 03:35:46 GMT+00:00 2006, Uncontrolled Copy, (c) BSI ISO 12080-2:2000(E) 4? 8.3? 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
43、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 liquid to a separating funnel (5.9) using two 30 ml portions of water, two 10 ml portions of e
44、thanol (4.1) and two 40 ml portions of light petroleum (4.5). Shake vigorously for 30 s and allow to stand until the two layers are clear. Transfer 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
45、separate. 8.3.4?Transfer the aqueous phase to a third separating funnel and the light petroleum phase to the first separating funnel. Wash the second separating funnel with two 10 ml portions of light petroleum (4.5). Add the washings to the first separating funnel. 8.3.5?Shake the aqueous phase wit
46、h 40 ml of light petroleum (4.5) and 10 ml of ethanol (4.1). Add the light petroleum phase to the first separating funnel. Wash the combined light petroleum extracts with three 40 ml portions of freshly prepared potassium hydroxide alcoholic solution (4.4), shaking vigorously. Then wash with 40 ml p
47、ortions of water until the last washing is neutral to phenolphthalein. Drain the last few drops of water, add two sheets of filter paper (5.11), cut into strips, to the separating funnel and shake. 8.3.6?Transfer the light petroleum extract, dried as described above, to a 200 ml one-mark volumetric
48、flask (5.5). Rinse the separating funnel and paper with light petroleum (4.5), add the rinsings to the volumetric flask and add 10 mg to 20 mg of BHT (4.9). Dilute with light petroleum to the 200 ml mark. 8.4? Preparation of test and reference solutions Pipette aliquot parts of the diluted extracts
49、(8.3.6) obtained from both the test solution (8.2) and the vitamin A standard solution (4.8) into separate round-bottom flasks. Evaporate to dryness under vacuum by swirling in a water bath (5.8) at a temperature not exceeding 40 C. Cool under running water and restore atmospheric pressure, preferably with nitrogen. Dissolve the residue immediately in 10,0 ml of methanol (4.6). 8.5? Determination Inject 20 ?l of the test solution and the reference solution (8.4) onto the column and adjust the operation conditions of the detector to give the