1、BSI Standards PublicationBS ISO 20633:2015Infant formula and adult nutritionals Determination of vitamin E and vitamin A by normal phase high performance liquid chromatographyBS ISO 20633:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 20633:2015.The UK pa
2、rticipation in its preparation was entrusted to Technical Committee AW/-/2, Food Technical Committee Chairmen.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Use
3、rs are responsible for its correct application. The British Standards Institution 2015. Published by BSI Standards Limited 2015ISBN 978 0 580 90424 0 ICS 67.050 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of
4、 the Standards Policy and Strategy Committee on 30 November 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 20633:2015 ISO 2015Infant formula and adult nutritionals Determination of vitamin E and vitamin A by normal phase high performance liquid chromatographyFo
5、rmules infantiles et produits nutritionnels pour adultes Dtermination de la teneur en vitamine E et de la teneur en vitamine A par chromatographie liquide haute performance en phase normaleINTERNATIONAL STANDARDISO20633First edition2015-11-01Reference numberISO 20633:2015(E)BS ISO 20633:2015ISO 2063
6、3:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in SwitzerlandAll rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, o
7、r posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax
8、+41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 20633:2015ISO 20633:2015(E)Foreword iv1 Scope . 12 Terms and definitions . 13 Principle 14 Reagents and materials . 25 Apparatus . 66 Procedure. 76.1 Sample preparation 76.1.1 General 76.1.2 Dry blended powder samples . 76.1.3 Wet blended powder samp
9、les 76.1.4 Liquid samples . 76.1.5 Sample extraction . 76.2 HPLC analysis . 76.2.1 General 76.2.2 Detector settings 86.2.3 Pump gradient elution cycle 87 System suitability. 88 Calculations 8Annex A (informative) Examples of chromatograms 10Annex B (informative) Precision data 16Annex C (informative
10、) Comparison between AOAC 2012.10, EN 12822 and EN 12823-1 19Bibliography .23 ISO 2015 All rights reserved iiiContents PageBS ISO 20633:2015ISO 20633:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). Th
11、e 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 be represented on that committee. International organizations, governmental and non-gov
12、ernmental, 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.The procedures used to develop this document and those intended for its further maintenance are described i
13、n the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).Attention is drawn to the p
14、ossibility 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. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO lis
15、t of patent declarations received (see www.iso.org/patents).Any trade name used in this document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well
16、as information about ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products in collaboration with AOAC INTERNATIONAL. It is being published by ISO
17、 and separately by AOAC INTERNATIONAL. The method described in this International Standard is equivalent to the AOAC Official Method 2012-10: Infant formula and adult nutritionals Determination of vitamin E and vitamin A by normal phase high performance liquid chromatography.iv ISO 2015 All rights r
18、eservedBS ISO 20633:2015INTERNATIONAL STANDARD ISO 20633:2015(E)Infant formula and adult nutritionals Determination of vitamin E and vitamin A by normal phase high performance liquid chromatographyWARNING The use of this International Standard can involve hazardous materials, operations and equipmen
19、t. This International Standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this International Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
20、1 ScopeThis International Standard specifies a method for the simultaneous quantitative determination of vitamin E (-tocopherol and -tocopheryl acetate) and vitamin A (13-cis and all-trans isomers of retinyl palmitate and retinyl acetate) present in all forms of infant and adult formulas (powders, r
21、eady-to-feed liquids and liquid concentrates).Retinol is not used for fortification purposes and therefore is not addressed in this method. The innate amount in products is insignificant.Stereoisomers of vitamin E, -tocopherol and -tocopheryl acetate, are not differentiated in this method.2 Terms an
22、d definitionsFor the purposes of this document, the following terms and definitions apply.2.1adult nutritionalnutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, made from any combination of milk, soy, rice, whey, hydrolyse
23、d protein, starch and amino acids, with and without intact protein2.2infant formulabreast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feedingSOURCE: Codex Sta
24、ndard 72-19813 PrincipleThis procedure utilizes the proteolytic enzyme, papain, to hydrolyze the hydrophilic protein coating of fat micelles in milk or soy-based infant formulations in an aqueous solution. The hydrophobic contents of the micelles are then extracted quantitatively into iso-octane in
25、a single extraction. The extract is analysed by normal phase HPLC using an analytical column with gradient elution. Quantification of -tocopherol and -tocopheryl acetate is done using fluorescence detection with excitation and emission wavelengths at 280 nm and 310 nm. Retinyl palmitate (cis and tra
26、ns) and retinyl acetate (cis and trans) are quantified using UV detection at 325 nm. ISO 2015 All rights reserved 1BS ISO 20633:2015ISO 20633:2015(E)4 Reagents and materialsDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized w
27、ater or water of equivalent purity.4.1 Methyl-t-butyl ether, also known as tert-butylmethylether, HPLC grade.4.2 n-Hexane, HPLC grade.4.3 Ethanol, HPLC grade.4.4 Methanol, HPLC grade.4.5 Iso-octane (2,2,4-trimethylpentane), HPLC grade.4.6 Papain (from Carica papaya), 3 U/mg, Sigma 762201)or equivale
28、nt.4.7 Hydroquinone, Sigma H90031)or equivalent.4.8 Glacial acetic acid, analytical reagent grade.4.9 Anhydrous sodium acetate.4.10 Dilute hydrochloric acid solution.Dilute 100 ml of a hydrochloric acid solution with a mass fraction of 36 % to 200 ml with water.4.11 Papain solution, mass concentrati
29、on = 20 g/l.Dissolve 100 mg hydroquinone and 4 g anhydrous sodium acetate in approximately 80 ml of water in a 100 ml one-mark volumetric flask (5.11). Adjust the pH to 5,0 with dilute hydrochloric acid solution (4.10). Add 2 g of papain and make up to volume. Prepare fresh prior to use.4.12 Acidifi
30、ed methanol solution.Add 20 ml of glacial acetic acid to 1 l of methanol and mix. Prepare fresh on the day of use.4.13 HPLC mobile phase A.n-Hexane, filtered and degassed for 15 min in an ultrasonic bath.4.14 HPLC mobile phase B.Mix 750 ml of n-hexane with 250 ml of methyl-t-butyl ether. Add 3 ml of
31、 methanol, filter and degas for 15 min in an ultrasonic bath.4.15 Standard substances4.15.1 Retinyl palmitate reference standard, primary reference standard. The standard shall contain antioxidant. CAS 78-81-2.1) This is an example of a suitable product available commercially. This information is gi
32、ven for the convenience of users of this document and does not constitute an endorsement by ISO of the product named. Equivalent products may be used if they can be shown to lead to the same results.2 ISO 2015 All rights reservedBS ISO 20633:2015ISO 20633:2015(E)4.15.2 Retinyl acetate reference stan
33、dard, primary reference standard. CAS 127-47-9.4.15.3 -tocopheryl acetate reference standard, primary reference standard. CAS 7695-91-24.15.4 -tocopherol reference standard, primary reference standard. CAS 10191-41-0.4.16 Standard solutions4.16.1 Retinyl palmitate stock standard solution.Weigh to th
34、e nearest 0,01 mg, approximately 70 mg of retinyl palmitate (4.15.1) into a 50 ml volumetric flask (5.11). Dissolve in and dilute to volume with iso-octane (4.5).4.16.2 Retinyl acetate stock standard solution.Weigh, to the nearest 0,01 mg, approximately 35 mg of retinyl acetate (4.15.2) into a 50 ml
35、 volumetric flask (5.11). Dissolve in and dilute to volume with ethanol (4.3).4.16.3 -tocopheryl acetate stock standard solution.Weigh, to the nearest 0,01 mg, approximately 180 mg of -tocopheryl acetate (4.15.3) into a 50 ml volumetric flask (5.11). Dissolve in and dilute to volume with iso-octane.
36、4.16.4 -tocopherol stock standard solution.Weigh, to the nearest 0,01 mg, approximately 100 mg of -tocopherol (4.15.4) into a 50 ml volumetric flask (5.11). Dissolve in and dilute to volume with iso-octane.NOTE The above stock standard solutions are stable in a refrigerator at 4 C to 8 C for up to 7
37、 days.4.16.5 Combined working standard solution 1.Transfer by pipette 4 ml of retinyl palmitate stock standard solution (4.16.1), 4 ml of retinyl acetate stock standard solution (4.16.2), 7 ml of -tocopheryl acetate stock standard solution (4.16.3) and 20 ml of -tocopherol stock standard solution (4
38、.16.4), into a 50 ml volumetric flask (5.11) and dilute to volume with iso-octane. Prepare this solution freshly prior to use.4.16.6 Combined working standard solution 2.Transfer by pipette 8 ml of combined working standard solution 1 (4.16.5) into a 100 ml volumetric flask (5.11) and dilute to volu
39、me with iso-octane. Prepare this solution freshly prior to use.4.16.7 Calibration standard solutionsInto separate 50 ml volumetric flasks (5.11), transfer by pipette 0,5 ml, 2 ml, 4 ml, 8 ml, 16 ml and 32 ml of combined working standard solution 2 (4.16.6), and dilute to volume with iso-octane. Thes
40、e solutions are used to construct a multipoint calibration curve. Prepare these solutions daily prior to use.NOTE For routine testing and depending on the concentration range of the analytes in the test samples, a 3 or 4 point standard curve can be used, provided the ranges are within the lowest and
41、 highest points of the 6 point curve listed above. ISO 2015 All rights reserved 3BS ISO 20633:2015ISO 20633:2015(E)4.17 Stock standard purity determinations4.17.1 Spectrometric purity of retinyl palmitate stock solutionPipet 1 ml of retinyl palmitate stock standard solution (4.16.1) into a 100 ml vo
42、lumetric flask (5.11) and make up to volume with ethanol. Determine the absorption at 325 nm, zeroed against ethanol in a 1 cm quartz cell. Repeat the reading a further two times, rinsing the sample cuvette with the solution before each reading. Calculate the average absorbance reading. Calculate th
43、e spectrometric purity as a decimal, SPRP, of retinyl palmitate using Formula (1):SPAmRPst=97550 100110 (1)whereA is the average absorbance reading;975 is the extinction coefficient of retinyl palmitate at 325 nm (see Reference 1);mstis the mass of the reference standard in mg.4.17.2 Spectrometric p
44、urity of retinyl acetate stock solutionPipet 1 ml of retinyl acetate stock standard solution (4.16.2), into a 100 ml volumetric flask (5.11) and make up to volume with ethanol. Determine the absorption at 325 nm, zeroed against ethanol in a 1 cm quartz cell. Repeat the reading a further two times, r
45、insing the sample cuvette with the solution before each reading. Calculate the average absorbance reading. Calculate the spectrometric purity as a decimal, SPRA, of retinyl acetate using Formula (2):SPAmRAst=156050 100110 (2)whereA is the average absorbance reading;1 560 is the extinction coefficien
46、t of retinyl acetate at 325 nm (see Reference 1);mstis the mass of the reference standard in mg.4.17.3 Spectrometric purity of -tocopheryl acetate stock solutionPipet 3 ml of -tocopheryl acetate stock standard solution (4.16.3), into a 100 ml volumetric flask (5.11) and make up to volume with ethano
47、l. Determine the absorption at 284 nm, zeroed against ethanol in a 1 cm quartz cell. Repeat the reading a further two times, rinsing the sample cuvette with the solution before each reading. Calculate the average absorbance reading. Calculate the spectrometric purity as a decimal, SPTA, of -tocopher
48、yl acetate using Formula (3):SPAmTAst=43 650 100310,(3)whereA is the average absorbance reading;43,6 is the extinction coefficient of -tocopheryl acetate at 284 nm (see Reference 1);mstis the mass of the reference standard in mg.4 ISO 2015 All rights reservedBS ISO 20633:2015ISO 20633:2015(E)4.17.4
49、Spectrometric purity of -tocopherol stock solutionPipet 3 ml of -tocopherol stock standard solution (4.16.4), into a 100 ml volumetric flask (5.11) and make up to volume with ethanol. Determine the absorption at 292 nm, zeroed against ethanol in a 1 cm quartz cell. Repeat the reading a further two times, rinsing the sample cuvette with the solution before each reading. Calculate the average absorbance reading. Calculate the spectrometric purity as a decimal, SPT, of -tocopherol using Formula (4):SPAmTst
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