1、BSI Standards PublicationBS ISO 20638:2015Infant formula Determination of nucleotidesby liquid chromatographyBS ISO 20638:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 20638:2015.The UK participation in its preparation was entrusted to TechnicalCommittee
2、 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 its correctapplication. The British Standards
3、Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 90421 9ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 November 2015.Amendments
4、/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 20638:2015 ISO 2015Infant formula Determination of nucleotides by liquid chromatographyFormules infantiles Dtermination de la teneur en nuclotides par chromatographie liquideINTERNATIONAL STANDARDISO20638First edition2015-11-01Re
5、ference numberISO 20638:2015(E)BS ISO 20638:2015ISO 20638:2015(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 me
6、ans, electronic or mechanical, including photocopying, or 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-12
7、14 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 20638:2015ISO 20638:2015(E)Foreword iv1 Scope . 12 Terms and definitions . 13 Principle 14 Reagents and materials . 15 Apparatus . 46 Sample preparation . 47 Procedure. 57.1 Extraction 57.2 Chro
8、matography 58 Calculations 69 Results . 8Annex A (informative) Examples of chromatograms . 9Annex B (informative) Precision data 10Bibliography .14 ISO 2015 All rights reserved iiiContents PageBS ISO 20638:2015ISO 20638:2015(E)ForewordISO (the International Organization for Standardization) is a wor
9、ldwide 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
10、 that 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 d
11、ocument 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 Direc
12、tives, Part 2. www.iso.org/directivesAttention 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 development o
13、f the document will be in the Introduction and/or on the ISO list of patent declarations received. www.iso.org/patentsAny 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
14、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 collabora
15、tion with AOAC INTERNATIONAL. It is being published by ISO and separately by AOAC INTERNATIONAL. The method described in this International Standard is equivalent to the AOAC Official Method 2011.20: Nucleotides in infant formula.iv ISO 2015 All rights reservedBS ISO 20638:2015INTERNATIONAL STANDARD
16、 ISO 20638:2015(E)Infant formula Determination of nucleotides by liquid chromatographyWARNING 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
17、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.1 ScopeThis International Standard specifies a method for the quantitative determination of 5-mononucleotides i
18、n infant formula in solid (i.e. powders) or liquid (i.e. ready-to-feed liquids and liquid concentrates) forms using liquid chromatography.2 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.2.1infant formulabreast-milk substitute specially manufactured
19、 to satisfy, 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 PrincipleThe sample is dissolved in high-salt solution to inhibit protein and fat interactions. The 5-mononucleot
20、ides uridine 5-monophosphate (UMP), inosine 5-monophosphate (IMP), adenosine 5-monophosphate (AMP), guanosine 5-monophosphate (GMP), and cytidine 5-monophosphate (CMP) are separated from the sample matrix by strong-anion exchange solid-phase extraction (SPE), followed by chromatographic analysis usi
21、ng a C18 stationary phase with gradient elution, UV detection, and quantitation by an internal standard technique using thymidine 5-monophosphate (TMP).14 Reagents and materialsDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and distilled or demineraliz
22、ed water or water of equivalent purity.4.1 Standards, 99 % pure (Sigma1)or equivalent). Nucleotide sodium salts or sodium salt hydrates may be substituted if free acid forms are not readily available.4.1.1 TMP, thymidine 5-monophosphate, CAS No. 365-07-1.4.1.2 AMP, adenosine 5-monophosphate, CAS No.
23、 61-19-8.4.1.3 CMP, cytidine 5-monophosphate, CAS No. 63-37-6.1) 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 use
24、d if they can be shown to lead to the same results. ISO 2015 All rights reserved 1BS ISO 20638:2015ISO 20638:2015(E)4.1.4 GMP, guanosine 5-monophosphate, CAS No. 85-32-5.4.1.5 IMP, inosine 5-monophosphate, CAS No. 131-99-7.4.1.6 UMP, uridine 5-monophosphate, CAS No. 58-97-9.4.2 Potassium bromide (KB
25、r).4.3 Potassium dihydrogen phosphate (KH2PO4).4.4 Orthophosphoric acid (H3PO4).4.5 Potassium hydroxide (KOH).4.6 Ethylenediaminetetraacetic acid, disodium salt dihydrate (EDTA).4.7 Sodium chloride (NaCl).4.8 Methanol (CH3OH).4.9 Reagent preparation4.9.1 Standardizing buffer (KH2PO4, c = 0,25 mol/l,
26、 pH = 3,5). Dissolve 34,0 g KH2PO4(4.3) in 900 ml water and adjust pH to 3,5 with orthophosphoric acid (4.4). Dilute to 1 l.4.9.2 Extraction solution (NaCl, c = 1 mol/l, EDTA c = 4 mmol/l). Dissolve 58,5 g NaCl (4.7) and 1,5 g EDTA (4.6). Dilute in 1 l water.4.9.3 Wash solution (KBr, c = 0,3 mol/l).
27、 Dissolve 3,6 g KBr (4.2) in 100 ml water.4.9.4 Eluent solution (KH2PO4, c = 0,5 mol/l, pH = 3,0). Dissolve 6,8 g KH2PO4(4.3) in 90 ml water and adjust pH to 3,0 with orthophosphoric acid (4.4). Dilute to 100 ml.4.9.5 Mobile phase A (KH2PO4, c = 10 mmol/l, pH = 5,6). Dissolve 1,4 g KH2PO4(4.3) in 90
28、0 ml water and adjust pH to 5,6 0,1 with KOH solution (10 % m/v). Dilute to 1 l with water. Make daily as microbial growth often occurs at room temperature in phosphate buffers that contain little or no organic solvent.4.9.6 Mobile phase B, 100 % methanol (4.8).4.10 Standard preparation4.10.1 Stock
29、standard solutions, approximately 1 mg/ml. Accurately weigh approximately 50 mg each nucleotide 5-monophosphate into separate 50 ml volumetric flasks. Add 40 ml water, mix until dissolved, and make to volume with water.4.10.2 Purity standard solutions. Pipette 1,0 ml each stock standard (4.10.1) int
30、o separate 50 ml volumetric flasks, make to volume with standardizing buffer (4.9.1), and measure absorbance at the appropriate maxto determine the concentration of each nucleotide stock standard. See Table 1 and References 1 and 2.2 ISO 2015 All rights reservedBS ISO 20638:2015ISO 20638:2015(E)Tabl
31、e 1 UV absorbance maxima and extinction coefficients for nucleotide 5-monophosphatesNucleotide 5-monophosphatemaxnmE11cm%Adenosine 5-monophosphate 257 428,6Cytidine 5-monophosphate 280 390,9Guanosine 5-monophosphate 254 392,0Inosine 5-monophosphate 249 356,5Uridine 5-monophosphate 262 312,7Thymidine
32、 5-monophosphate 267 288,54.10.3 Internal standard solution, approximately 80 g/ml. Dilute 4 ml TMP stock standard (4.10.1) into 50 ml water.4.10.4 Working standard solution, approximately 40 g/ml. Pipette 2 ml each stock standard (4.10.1) (AMP, CMP, GMP, IMP, and UMP) into a single 50 ml volumetric
33、 flask and make to volume with water.4.10.5 Calibration standard solutions. See Table 2 for nominal nucleotide concentrations of the calibration standard solutions.4.10.5.1 Calibration standard 1. Pipette 0,25 ml working standard (4.10.4) and 1 ml internal standard (4.10.3) into a 25 ml volumetric f
34、lask and make to volume with water.4.10.5.2 Calibration standard 2. Pipette 0,5 ml working standard (4.10.4) and 1 ml internal standard (4.10.3) into a 25 ml volumetric flask and make to volume with water.4.10.5.3 Calibration standard 3. Pipette 2 ml working standard (4.10.4) and 1 ml internal stand
35、ard (4.10.3) into a 25 ml volumetric flask and make to volume with water.4.10.5.4 Calibration standard 4. Pipette 5 ml working standard (4.10.4) and 1 ml internal standard (4.10.3) into a 25 ml volumetric flask and make to volume with water.Table 2 Nominal concentration of calibration standardsCalib
36、ration solutionConcentration of each nucleotide: AMP, CMP, GMP,IMP, UMP g/mlConcentration of TMP g/ml1 0,4 3,22 0,8 3,23 3,2 3,24 8,0 3,2 ISO 2015 All rights reserved 3BS ISO 20638:2015ISO 20638:2015(E)5 ApparatusUsual laboratory glassware and equipment and, in particular, the following.5.1 HPLC sys
37、tem, equipped with pump, sample injector unit with a 50 l injection loop, degasser unit, column oven, and photodiode array detector.5.2 C18 column, Gemini2)C18, 5 m, 4,6 mm 250 mm (Phenomenex2).5.3 Spectrophotometer, capable of digital readout to 3 decimal places.5.4 pH-meter.5.5 Centrifuge.5.6 Cent
38、rifuge tubes, Amicon Ultra MWCO 3k, 4 ml (Millipore)2).5.7 Polypropylene centrifuge tubes, capacity 50 ml.5.8 Disposable syringes, capacity 3 ml.5.9 Syringe filters, 0,2 m with cellulose acetate membranes.5.10 SPE vacuum manifold.5.11 Polypropylene strong-anion exchange SPE cartridges, 6 ml 1 000 mg
39、, Chromabond SB2).5.12 Filter membranes, 0,45 m nylon.6 Sample preparationa) Shake or mix sample container prior to opening.b) Accurately weigh approximately 1 g powder or 10 ml ready-to-feed/liquid milk infant formula into a 50 ml centrifuge tube.c) Add 30 ml extraction solution (4.9.2).d) Add 1,0
40、ml TMP internal standard (4.10.3).e) Cap the tube and vortex mix until powder dissolved.f) Allow sample to stand for 10 min to ensure complete hydration.g) Dilute to a final volume of approximately 50 ml with water.h) Cap the tube and vortex mix.i) For starch based products, transfer 2 4 ml of prepa
41、red sample to two separate ultra centrifuge tubes and centrifuge at 3 500g for 60 min, then pool filtrate from both tubes.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
42、 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 20638:2015ISO 20638:2015(E)7 Procedure7.1 ExtractionThroughout the extraction procedure, do not let the cartridge run dry but drain to the top of the ca
43、rtridge bed only.When draining the cartridge, the flow rate should be 2 ml/min.a) For each sample, place a single SPE cartridge on a vacuum manifold.b) Condition the columns by adding with 4 ml methanol and draining to top of the cartridge bed; followed by adding 2 lots of water (5 ml each) and drai
44、ning to top of cartridge bed.c) Load the cartridge with 4 ml of sample solution and drain to the top of the cartridge bed.d) Wash the cartridge to remove interferences with 4 ml of wash solution (4.9.3) and drain to the top of the cartridge bed.e) Place a sample collection tube in SPE manifold.f) El
45、ute the nucleotides with 4 ml of eluent solution (4.9.4) into a sample collection tube and completely drain the cartridge.g) Filter an aliquot of approximately 2 ml of the eluent through a 0,2 m syringe filter into an autosampler vial.7.2 Chromatographya) Form gradients by low pressure mixing of the
46、 two mobile phases, A and B, with separation of nucleotides achieved using the procedure given in Table 3.Table 3 Gradient procedure for chromatographic separationTimeminFlow rateml/minMobile Phase A%Mobile Phase B%0 0,6 100 025 0,6 80 2026 0,6 100 040 0,6 100 0b) Acquire spectral data between 210 n
47、m and 300 nm by the photodiode array detector with chromatograms monitored at the specified following wavelengths for quantitation.1) IMP: wavelength at 250 nm.2) AMP, GMP, and TMP: wavelength at 260 nm.3) CMP and UMP: wavelength at 270 nm.c) Set column oven to 40 C.Example chromatograms can be foun
48、d in Annex A. ISO 2015 All rights reserved 5BS ISO 20638:2015ISO 20638:2015(E)8 Calculations8.1 Calculate the percent purity of each nucleotide (as free acid) in purity standard solution (4.10.2), using Formula (1):Purity,%AbsESS 1max1cm1%=50 501 000m(1)whereAbsmaxis the UV absorbance at maximum wav
49、elength;E1cm1%is the extinction coefficient for nucleotide;mSS is the mass of nucleotide in stock standard (mg);50 is the total volume of stock standard (ml);50 is the total volume of purity standard (ml);1 is the volume of stock standard added to purity standard (ml);1 000 is the mass conversion from mg to g.8.2 Calculate the concentration of nucleotide in stock standard solution (SS) (4.10.1), using Formula (2):SS (g/ml)=mSS PS%50 100103(2)wheremSS is the mass of nucle
