1、BSI Standards PublicationBS ISO 20637:2015Infant formula and adultnutritionals Determinationof myo-inositol by liquidchromatography and pulsedamperometryBS ISO 20637:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 20637:2015.The UK participation in its pre
2、paration was entrusted to TechnicalCommittee AW/-/2, Food Technical Committee Chairmen.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for it
3、s correctapplication. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 90420 2ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strat
4、egy Committee on 30 November 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 20637:2015 ISO 2015Infant formula and adult nutritionals Determination of myo-inositol by liquid chromatography and pulsed amperometryFormules infantiles et produits nutritionnels pour
5、adultes Dtermination de la teneur en myo-inositol par chromatographie liquide et ampromtrie pulseINTERNATIONAL STANDARDISO20637First edition2015-11-01Reference numberISO 20637:2015(E)BS ISO 20637:2015ISO 20637:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in
6、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 internet or an intranet, without prior written permission. Permission can b
7、e 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 47copyrightiso.orgwww.iso.orgBS ISO 20637:2015ISO 20637:2015(E)Foreword iv1 Sc
8、ope . 12 Terms and definitions . 13 Principle 14 Reagents and materials . 15 Apparatus . 36 Procedure. 46.1 Free myo-inositol 46.1.1 Sample preparation . 46.1.2 Extraction . 56.2 Myo-inositol bound as phosphatidylinositol . 56.2.1 Sample preparation . 56.2.2 Extraction . 56.2.3 Cleanup 66.2.4 Hydrol
9、ysis 66.3 HPLC analysis . 66.3.1 Instrument operating conditions 66.3.2 PAD settings with gold electrode . 86.3.3 Instrument startup. 86.3.4 Standard and sample analysis 86.3.5 System shutdown 87 Calculations 87.1 General . 87.2 Concentration of calibration standards . 97.3 Preparation of standard c
10、urve . 97.4 Calculation of free or free plus bound myo-inositol in samples . 97.4.1 Calculation of free myo-inositol 97.4.2 Calculation of bound myo-inositol 107.4.3 Calculation of free plus bound myo-inositol .10Annex A (informative) Examples of chromatograms 11Annex B (informative) Precision data
11、12Bibliography .15 ISO 2015 All rights reserved iiiContents PageBS ISO 20637:2015ISO 20637:2015(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally car
12、ried 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 in the work.
13、 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 in the ISO/IEC Directives, Part 1. In particular the differe
14、nt 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. www.iso.org/directivesAttention is drawn to the possibility that some of the elements of this document may be the
15、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 list of patent declarations received. www.iso.org/patentsAny trade n
16、ame 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 as information about ISOs adherence to the WTO principles in the Techni
17、cal 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 and separately by AOAC INTERNATIONAL. The method described in this Int
18、ernational Standard is equivalent to the AOAC Official Method 2011.18: Myo-inositol (free and bound as phosphatidyl inositol) in infant and pediatric formula and adult nutritional.iv ISO 2015 All rights reservedBS ISO 20637:2015INTERNATIONAL STANDARD ISO 20637:2015(E)Infant formula and adult nutriti
19、onals Determination of myo-inositol by liquid chromatography and pulsed amperometryWARNING The use of this International Standard can involve hazardous materials, operations and equipment. This International Standard does not purport to address all the safety problems associated with its use. It is
20、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.1 ScopeThis International Standard specifies a method for the determination of myo-inositol (free or free plus bou
21、nd as phosphatidylinositol) in infant formula and adult nutritionals using liquid chromatography and pulsed amperometry with column switching.2 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.2.1adult nutritionalnutritionally complete, specially form
22、ulated 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 protein2.2infant formulabreast-milk substitute specially manufactured to satisfy, by itse
23、lf, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feedingSOURCE: Codex Standard 72-19813 PrincipleFree myo-inositol and phosphatidyl bound myo-inositol are extracted using two different sample preparation procedures. Free
24、myo-inositol is extracted from samples with dilute hydrochloric acid and water. Phosphatidylinositol is extracted from samples with chloroform and separated from other fats with silica solid phase extraction cartridges. Myo-inositol is then released from the glycerol backbone with concentrated aceti
25、c and hydrochloric acid at 120C. The ion chromatographic method uses a combination of two different ion exchange columns with column switching and pulsed amperometric detection (PAD). The concentration of myo-inositol is calculated by comparison with external standards of known concentration.4 Reage
26、nts 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 20637:2015ISO 20637:2015(E)4.1 Chemicals and solvents4.1.1 Acetic acid, glacial, AC
27、S.4.1.2 Chloroform, high-purity, HPLC grade.4.1.3 Diethyl ether, anhydrous, HPLC grade.4.1.4 Drierite, (desiccant), anhydrous calcium sulfate, 8 mesh.4.1.5 Helium, zero grade or equivalent.4.1.6 Hexane, HPLC grade.4.1.7 Hydrochloric acid, concentrated (36 % to 38 %), ACS.4.1.8 Metaphosphoric acid, A
28、CS.4.1.9 Methanol, HPLC grade.4.1.10 Myo-inositol, primary reference standard, official lot, store desiccated. See standard label for purity.4.1.11 Sodium chloride, ACS.4.1.12 Sodium hydroxide, 50 % (m/m), low carbonate form.4.2 Preparation of reagents and standard solutions4.2.1 General. All soluti
29、ons can be scaled up or down for convenience provided good laboratory practices are observed. Solutions can be stored at refrigerated or at ambient temperature in tight, inert containers unless otherwise specified.4.2.2 Myo-inositol stock standard solution (approximately 2 000 mg/l). Accurately weig
30、h approximately 0,100 g myo-inositol and quantitatively transfer to a 50 ml volumetric flask. Dilute to volume with water. Mix well. Store refrigerated. Expiration: 3 months.4.2.3 Myo-inositol intermediate standard solution (approximately 200 mg/l). Dilute 10,0 ml stock standard (4.2.2) to 100 ml wi
31、th water and mix well. Discard after use.4.2.4 Preparation of calibration standard solutions4.2.4.1 Myo-inositol calibration standard solutions high, (approximately 4 mg/l, 2 mg/l, 1 mg/l, 0,5 mg/l).Into separate volumetric flasks, dilute 2,0 ml, 1,0 ml and 0,5 ml myo-inositol intermediate standard
32、(4.2.3) to 100 ml with water. Dilute 0,5 ml myo-inositol intermediate standard (4.2.3) to 200 ml with water. Expiration: 2 weeks4.2.4.2 Myo-inositol calibration standard solutions low, (approximately 0,2 mg/l and 0,05 mg/l).Into separate volumetric flasks, dilute 4 ml and 1 ml of the 0,5 mg/l myo-in
33、ositol calibration standard to 10 ml with water. Expiration: 2 weeks.2 ISO 2015 All rights reservedBS ISO 20637:2015ISO 20637:2015(E)4.2.5 Hydrochloric acid, 0,5 %. Add 1,25 ml concentrated hydrochloric acid to approximately 200 ml water in a 250 ml volumetric flask. Dilute to volume with water and
34、mix well. Expiration: 6 months.4.2.6 Sodium chloride, 1 mol/l. Dissolve 5,8 g sodium chloride and dilute to 100 ml with water. Expiration: 1 month.4.2.7 Sodium hydroxide, 0,12 % or 30 mmol (Pump 1). Quickly weigh (4,8 0,1) g of 50 % sodium hydroxide into a 2 000 ml volumetric flask containing approx
35、imately 1 900 ml water. It is important that the sodium hydroxide does not absorb carbon dioxide from the air. Swirl to mix well. Dilute to volume with water and mix well. Expiration: 1 month.4.2.8 Sodium hydroxide, 4,0 % or 1 mol/l (Pump 2). Quickly weigh (160 3) g of 50 % sodium hydroxide into a 2
36、 000 ml volumetric flask containing approximately 1 900 ml water. It is important that the sodium hydroxide does not absorb carbon dioxide from the air. Swirl to mix well. Dilute to volume with water and mix well. Expiration: 1 month.4.2.9 Metaphosphoric acid, 6 %. Weigh 6,0 g metaphosphoric acid in
37、to a 100 ml volumetric flask. Dissolve and dilute to volume with water. Mix well. Store refrigerated. Expiration: 1 week.4.2.10 Phosphatidylinositol extraction solutions. Prepare fresh on day of use.4.2.10.1 Chloroform:methanol (2:1). Mix 60 ml chloroform and 30 ml methanol.4.2.10.2 Hexane:diethyl e
38、ther (80:20). Mix 80 ml hexane and 20 ml diethyl ether.4.2.10.3 Hexane:diethyl ether (50:50). Mix 50 ml hexane and 50 ml diethyl ether.4.2.10.4 Methanol:chloroform:water (75:15:10). Mix 75 ml methanol, 15 ml chloroform and 10 ml water.5 ApparatusUsual laboratory glassware and equipment and, in parti
39、cular, the following.5.1 Analytical balance, minimum weighing capacity of at least 0,000 1 g.5.2 Centrifuge.5.3 Desiccator.5.4 Nitrogen evaporator, with water bath or equivalent.5.5 Oven, capable of maintaining 120 C.5.6 pH-meter, with pH 4 and 7 buffers.5.7 Stir plate, multiposition with stir bars.
40、5.8 Vacuum manifold.5.9 Vortex mixer. ISO 2015 All rights reserved 3BS ISO 20637:2015ISO 20637:2015(E)5.10 System HPLC, with corrosion-resistant components including an autosampler, two isocratic pumps, 6-port switching valve, pulsed amperometry detector with a gold electrode and PEEK or polytetrafl
41、uoroethylene (PTFE) 0,18 mm to 0,25 mm (0,007 inch to 0,01 inch) internal diameter tubing. Autosampler capable of injecting 20 l.5.11 Columns, Dionex CarboPac1)MA1 (4 mm 250 mm) P/N, 44066, MA1 (4 mm 50 mm) P/N 44067, and PA1 (4 50 mm) P/N 43096, or equivalent.5.12 Beakers, assorted sizes.5.13 Centr
42、ifuge tubes, 50 ml with polytetrafluoroethylene (PTFE)-coated caps.5.14 Syringe filters, polyamide, 0,45 m and 0,2 m.5.15 Filter paper, Whatman 2V1)or equivalent.5.16 Conical flasks, 50 ml or 125 ml or equivalent.5.17 Volumetric flasks, assorted sizes.5.18 Funnels, suitable for use with filter paper
43、.5.19 Pipets, volumetric, assorted sizes.5.20 Solid-phase extraction (SPE) cartridge, silica, 1 g2).5.21 Syringes, 1 ml disposable and 25 ml gas-tight glass with 100 mm (4 in) stainless steel needles.6 Procedure6.1 Free myo-inositol6.1.1 Sample preparation6.1.1.1 GeneralPrepared samples that are con
44、stantly stored at 1 C to 8 C in closed containers are stable for up to 5 days. After 5 days, samples shall be prepared again. Mix liquid samples well to ensure homogeneity. If the powder sample homogeneity is unknown, assume that it is non-homogenous and proceed with the preparation of dry blended/n
45、on-homogenous powder samples as given in 6.1.1.3.6.1.1.2 Liquid samplesFor ready-to-feed liquid samples, accurately weigh (0,5 0,05) g to (5 0,5) g of product into a 100 ml volumetric flask and record the mass to the nearest 0,000 1 g.1) This is an example of a suitable product available commerciall
46、y. 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 same results.2) J.T. Baker P/N 7086-07 () is an example of a suitable product available c
47、ommercially. 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 same results.4 ISO 2015 All rights reservedBS ISO 20637:2015ISO 20637:2015(E)6.
48、1.1.3 Dry blended powder samplesFor dry blended/non-homogenous powder samples, reconstitute per the product label instructions. Accurately weigh 0,5 g to 5 g reconstituted product into a 100 ml volumetric flask. Record the mass to the nearest 0,000 1 g.6.1.1.4 Wet blended powder samplesFor wet blend
49、ed/homogenous powder samples, accurately weigh 0,25 g to 1,5 g powder into a 100 ml volumetric flask and record the mass to the nearest 0,000 1 g. Add approximately 10 ml to 15 ml water to the volumetric flask and swirl or stir to completely dissolve the powder.6.1.2 ExtractionAdd enough 0,5 % hydrochloric acid (4.2.5) to each sample to adjust the sample pH to 4,5 0,2 and swirl to mix.Allow the samples to react with 0,5 % hydrochloric acid for a minimum of 2 min and then dilute to volume with water.
