1、BSI Standards PublicationBS ISO 20634:2015Infant formula and adult nutritionals Determination of vitamin B12by reversed phase high performance liquid chromatography (RP-HPLC)BS ISO 20634:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 20634:2015.The UK par
2、ticipation 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. User
3、s are responsible for its correct application. The British Standards Institution 2015. Published by BSI Standards Limited 2015ISBN 978 0 580 90425 7 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 20634:2015 ISO 2015Infant formula and adult nutritionals Determination of vitamin B12 by reversed phase high performance liquid chromatography (RP-HPLC)For
5、mules infantiles et produits nutritionnels pour adultes Dtermination de la teneur en vitamine B12 par chromatographie liquide haute performance en phase inverse (CLHP-PI)INTERNATIONAL STANDARDISO20634First edition2015-11-01Reference numberISO 20634:2015(E)BS ISO 20634:2015ISO 20634:2015(E)ii ISO 201
6、5 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, or posting on the in
7、ternet 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 +41 22 749 09 47cop
8、yrightiso.orgwww.iso.orgBS ISO 20634:2015ISO 20634:2015(E)Foreword iv1 Scope . 12 Terms and definitions . 13 Principle 14 Reagents and materials . 15 Apparatus . 46 Procedure. 56.1 General . 56.2 Sample preparation 56.2.1 General 56.2.2 Extraction . 66.2.3 Sample concentration 66.3 HPLC analysis . 7
9、6.3.1 System setup and configuration . 76.3.2 Instrument operation conditions 77 Calculations 87.1 General . 87.2 Calculation of standard solution concentrations 97.3 Preparation of standard curves 97.4 Calculation of vitamin B12concentrations in sample solutions . 9Annex A (informative) Examples of
10、 chromatograms 10Annex B (informative) Precision data 11Annex C (informative) Example of SPE cartridge qualification procedure 13Bibliography .14 ISO 2015 All rights reserved iiiContents PageBS ISO 20634:2015ISO 20634:2015(E)ForewordISO (the International Organization for Standardization) is a world
11、wide 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 be represented on t
12、hat 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.The procedures used to develop this doc
13、ument and those intended for its further maintenance are described in 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 Directi
14、ves, Part 2 (see www.iso.org/directives).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. Details of any patent rights identified during the developme
15、nt of the document will be in the Introduction and/or on the ISO list 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 speci
16、fic terms and expressions related to conformity assessment, as well 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
17、 collaboration with AOAC INTERNATIONAL. It is being published by ISO and separately by AOAC INTERNATIONAL. The method described in the International Standard is equivalent to the AOAC Official Method 2011.10: Vitamin B12in infant and pediatric formulas and adult nutritionals.iv ISO 2015 All rights r
18、eservedBS ISO 20634:2015INTERNATIONAL STANDARD ISO 20634:2015(E)Infant formula and adult nutritionals Determination of vitamin B12 by reversed phase high performance liquid chromatography (RP-HPLC)WARNING 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 quantitative determination of vitamin B12in infant and adult formula (powders, ready-to-feed liquids and liquid concentrates) by reversed phase high performance liquid chromatography.2 Terms and definitionsFor the purposes of this documen
21、t, the following terms and definitions apply.2.1vitamin B12cyanocobalamin and other cobalt-containing corrinoids with vitamin B12biological activity, such as aquocobalamin, hydroxycobalamin, methylcobalamin and adenosylcobalamin, converted to cyanocobalamin2.2adult nutritionalnutritionally complete,
22、 specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, made from any combination of milk, soy, rice, whey, hydrolysed protein, starch and amino acids, with and without intact protein2.3infant formulabreast-milk substitute specially manufactured to s
23、atisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feedingSOURCE: Codex Standard 72-19813 PrincipleCyanocobalamin and other cobalt-containing corrinoids are extracted from the sample using sodium acetate buff
24、er (pH = 4,5) and the latter converted to cyanocobalamin using potassium cyanide at 105 C. Extracts are purified and concentrated with C8 or C18 solid-phase extraction (SPE) cartridges and analysed with size-exclusion and reversed-phase chromatography. Determination of vitamin B12is made by liquid c
25、hromatography with visible detection at 550 nm.4 Reagents and materialsDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineralized water or water of equivalent purity. ISO 2015 All rights reserved 1BS ISO 20634:2015ISO 20634:2015(E)4.
26、1 Glacial acetic acid.4.2 Acetonitrile, HPLC grade.4.3 Drierite, desiccant, anhydrous calcium sulfate, 8 mesh.4.4 Ethanol, denatured.4.5 Formic acid, 88 %.4.6 Potassium cyanide, 97 %.4.7 Riboflavin, 98 % to 102 % purity.4.8 Sodium acetate anhydrous or sodium acetate trihydrate, ACS.4.9 Taka-Diastase
27、, Accurate Chemical Co.1)or equivalent.4.10 Triethylamine, HPLC grade.4.11 Vitamin B12(cyanocobalamin) primary reference standard, e.g. USP Reference 1152009 (approximately 10 g/mg), Official lot1). Store in a desiccator protected from white light.4.12 Preparation of solutions and standard solutions
28、4.12.1 GeneralAll solutions may be scaled up or down for convenience provided good laboratory practices are observed. Solutions can be stored refrigerated or at ambient temperature in tight, inert containers unless otherwise specified.4.12.2 Preparation of solutions4.12.2.1 HPLC mobile phase A. Dilu
29、te 4,0 ml of triethylamine with 1 000 ml of water. Adjust the pH to 5 to 7 with approximately 1,25 ml concentrated formic acid (4.5). Expiration: 1 week.4.12.2.2 HPLC mobile phase B. Mix 4,0 ml of triethylamine and 250 ml of acetonitrile with 750 ml of water. Adjust the pH to 5 to 7 with approximate
30、ly 1,25 ml concentrated formic acid. Expiration: 1 week in tightly stoppered container.4.12.2.3 HPLC mobile phase C. Mix 4,0 ml of triethylamine and 750 ml of acetonitrile with 250 ml of water. Adjust the pH to 5 to 7 with approximately 1,25 ml concentrated formic acid. Expiration: 1 week in tightly
31、 stoppered container.4.12.2.4 HPLC mobile phase D. Dilute 50 ml of acetonitrile to 2 000 ml with water. Expiration: 1 week in tightly stoppered container.4.12.2.5 Mixture of acetonitrile and water, volume fraction 10 %. Dilute 150 ml of acetonitrile to 1 500 ml with water. Expiration: 1 month in tig
32、htly stoppered container.1) This is an example of a suitable product available commercially. This information is given 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
33、 same results.2 ISO 2015 All rights reservedBS ISO 20634:2015ISO 20634:2015(E)4.12.2.6 Mixture of acetonitrile and water, SPE elution solvent, volume fraction 30 %. Dilute 30 ml of acetonitrile to 100 ml with water. Expiration: 1 month in tightly stoppered container.4.12.2.7 Mixture of acetonitrile
34、and water, column cleaning and storage solution, volume fraction 50 %. Dilute 500 ml of acetonitrile to 1 000 ml with water in a volumetric flask. Expiration: 6 months.4.12.2.8 Mixture of ethanol and water, volume fraction 25 %. Dilute 50 ml of ethanol to 200 ml with water. Expiration: 1 year in tig
35、htly stoppered container.4.12.2.9 Potassium cyanide solution, mass concentration = 4 g/l. Dissolve 0,02 g of potassium cyanide in and dilute to 5 ml with sodium acetate buffer (4.12.2.11) substance concentration c = 0,25 mol/l). Prepare fresh immediately before use.4.12.2.10 Potassium cyanide soluti
36、on, = 10 g/l. Dissolve 0,25 g of potassium cyanide in water and dilute to 25 ml. Prepare fresh immediately before use.4.12.2.11 Sodium acetate buffer, c = 0,25 mol/l. Dissolve 41,0 g of sodium acetate anhydrous or 68,0 g of sodium acetate trihydrate in approximately 1 800 ml of water. Adjust the pH
37、to 4,5 with concentrated acetic acid (approximately 40 ml). Dilute to 2 000 ml with water. Expiration: 3 months.4.12.2.12 Resolution test solution. Weigh approximately 0,005 g of riboflavin onto a weigh paper. Transfer to a 100 ml volumetric flask and bring to volume with 10 % acetonitrile solution
38、Stir to dissolve. Mix equal amounts of solution with the highest concentration of vitamin B12working standard solution. Expiration: 1 week.4.12.2.13 Taka-Diastase solution, = 60 g/l. Dissolve 0,6 g of Taka-Diastase in 10 ml of water. Prepare fresh daily before use.4.12.3 Preparation of standard solu
39、tions4.12.3.1 General. Prepare all standard solutions under UV shielded fluorescent lights and store at 2 C to 8 C in tightly stoppered volumetric flasks.4.12.3.2 Vitamin B12stock standard solution, = 10 000 g/l. Accurately weigh the appropriate amount of vitamin B12standard (4.11) to give a stock s
40、tandard concentration of 10 000 g/l. Dissolve in and dilute to 100 ml with 25 % ethanol (4.12.2.8). Expiration: 6 months.Calculate the amount of vitamin B12standard to be weighed, mw, in milligrams using Formula (1):mPw=10 000 011, (1)where10 000 is the desired stock standard solution concentration,
41、 in g/l;0,1 is the dilution volume, in l;P is the purity of the vitamin B12standard (4.11) in g of cyanocobalamin per mg of standard.4.12.3.3 Vitamin B12intermediate standard solution, = 1 000 g/l. Dilute 10 ml of stock standard solution (4.12.3.2) to 100 ml with water. Expiration: 1 week. ISO 2015
42、All rights reserved 3BS ISO 20634:2015ISO 20634:2015(E)4.12.3.4 Vitamin B12calibration standard solutions, = 2,5 g/l to 25 g/l. Into separate volumetric flasks, dilute 0,5 ml, 1 ml, 2 ml, 3 ml, 4 ml and 5 ml of intermediate standard solution (4.12.3.3) to 200 ml with 10 % acetonitrile (4.12.2.5). Ex
43、piration: 1 month.5 ApparatusUsual laboratory glassware and equipment and, in particular, the following.5.1 HPLC system, consisting of a gradient pump, column switching valve and isocratic pump, UV-VIS detector equipped with a tungsten lamp capable of monitoring at 550 nm and autosampler capable of
44、injecting 900 l to 2 000 l of sample.5.2 HPLC column, analytical size exclusion column: 4 m particle size, 250 mm 9,4 mm (e.g. Zorbax GF-2502)P/N 884973-901); 5 m, 300 mm 8 mm (Shodex Protein KW-802.52), P/N F6989000), or equivalent.5.3 HPLC column, analytical C18 column: 3 m particle size, 100 mm 4
45、,6 mm (e.g. Thermo Scientific Aquasil2)P/N 77503-104630) with C18 drop-in guard cartridges 3 m, 10 mm 4,6 mm (e.g. Thermo Scientific Aquasil2)P/N 77503-014001); Epic Phenyl Hexyl, 3 m, 120 , 100 mm 4,6 mm, (ES Industries 125191-EPHX)2)with appropriate guard cartridge, or equivalent reversed-phase co
46、lumn compatible with 100 % aqueous mobile phase).5.4 Oven, capable of maintaining temperatures of 95 C 5 C and 105 C 5 C.5.5 pH-meter, with calibration buffer.5.6 Analytical balance, accuracy to the nearest 0,000 01 g.5.7 Beakers, glass, assorted sizes.5.8 Bottle top dispenser, capable of dispensing
47、 30 ml or equivalent.5.9 Cylinders, graduated, glass, assorted sizes.5.10 Desiccator.5.11 Conical flasks, 125 ml capacity or equivalent glassware.5.12 Filter paper, Whatman 2V2)or equivalent.5.13 Funnels, plastic, suitable to use with filter paper.5.14 Gloves, disposable.5.15 Pipettor, variable volu
48、me, 100 l to 1 000 l.2) This is an example of a suitable product available commercially. This information is given 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 sam
49、e results.4 ISO 2015 All rights reservedBS ISO 20634:2015ISO 20634:2015(E)5.16 Laboratory light shields, yellow or clear shields with a cut off of at least 385 nm.5.17 Solid phase extraction (SPE) cartridges, C8, 900 mg (e.g. Alltech/Grace Davidson P/N 209662), C18, 900 mg (e.g. Alltech/Grace Davidson P/N 209422), or equivalent. An example of a SPE cartridge qualification procedure is given in Annex C.5.18 Syringes, disposable, assorted sizes.5.19 Syringe filters, 0,45 m nylon.5.20 Vacu
