1、BSI Standards PublicationBS ISO 20639:2015Infant formula and adult nutritionals Determination of pantothenic acid by ultra high performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS)BS ISO 20639:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implem
2、entation of ISO 20639:2015.The UK participation 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 nec
3、essary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2015. Published by BSI Standards Limited 2015ISBN 978 0 580 90422 6 ICS 67.050 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard
4、was published under the authority of 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 20639:2015 ISO 2015Infant formula and adult nutritionals Determination of pantothenic acid by ultra high performance l
5、iquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS)Formules infantiles et produits nutritionnels pour adultes Dtermination de la teneur en acide pantothnique par chromatographie liquide ultra haute performance et spectromtrie de masse en tandem (CLUHP-SM/SM)INTERNATIONAL STANDARDI
6、SO20639First edition2015-11-01Reference numberISO 20639:2015(E)BS ISO 20639:2015ISO 20639:2015(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 ot
7、herwise 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 be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright offic
8、eCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 20639:2015ISO 20639:2015(E)Foreword iv1 Scope . 12 Terms and definitions . 13 Principle 14 Reagents and materials . 15 Apparatus . 36 Procedure. 36.1 Sample prepa
9、ration 36.1.1 General 36.1.2 Dry blended powder samples . 36.1.3 Wet blended powder samples 46.1.4 Liquid samples . 46.2 Extraction 46.3 Analysis 46.3.1 Chromatographic analysis . 46.3.2 UHPLC conditions . 46.3.3 MS/MS conditions. 56.3.4 Identification . 57 Calculations 5Annex A (informative) Exampl
10、es of chromatograms . 6Annex B (informative) Precision data . 7Bibliography 8 ISO 2015 All rights reserved iiiContents PageBS ISO 20639:2015ISO 20639:2015(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. www.iso.org/directivesAttention is drawn to the possibi
14、lity 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 list of p
15、atent 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 and expressions related to conformity assessment, as well as informati
16、on 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 and separat
17、ely by AOAC INTERNATIONAL. The method described in this International Standard is equivalent to the AOAC Official Method 2012.16: Pantothenic acid (vitamin B5) in infant formula and adult/pediatric nutritional formula ultra high pressure liquid chromatography tandem mass spectrometry method.iv ISO 2
18、015 All rights reservedBS ISO 20639:2015INTERNATIONAL STANDARD ISO 20639:2015(E)Infant formula and adult nutritionals Determination of pantothenic acid by ultra high performance liquid chromatography and tandem mass spectrometry method (UHPLC-MS/MS)WARNING The use of this International Standard can
19、involve hazardous materials, operations and equipment. 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 a
20、pplicability of regulatory limitations prior to use.1 ScopeThis International Standard specifies a method for the quantitative determination of pantothenic acid, excluding bound forms, in infant formula and adult nutritionals (i.e. powders) using ultra high performance liquid chromatography and tand
21、em mass spectrometry method (UHPLC-MS/MS).2 Terms and 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
22、 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 itself, the nutritional requirements of infants during the first months of life up to the introduction o
23、f appropriate complementary feedingSOURCE: Codex Standard 72-19813 PrinciplePantothenic acid is extracted using a 0,4 mol/l ammonium acetate buffer solution. After filtration, the final solution is subjected to ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS).4 Rea
24、gents 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 20639:2015ISO 20639:2015(E)4.1 Standards4.1.1 Calcium d-pantothenate, Sigma1)or e
25、quivalent CAS 137-08-6.4.1.2 Calcium pantothenate-13C6, 15N2, IsoSciences1)or equivalent CAS 356786-94-2.4.2 -Amylase, Sigma A31761), from porcine pancreas, about 25 U/mg or equivalent.4.3 Solvents4.3.1 Acetonitrile, LC grade or equivalent.4.4 Ammonium acetate, ACS grade, 98 % (Fluka 9690)1).4.5 Ace
26、tic acid, ACS grade.4.6 Formic acid, ACS grade.4.7 1 % Formic acid in water, ACS grade.4.8 Preparation of standard solutions4.8.1 Pantothenic acid (PA) stock solution, = 250 g/ml. Weigh 54,5 mg of calcium pantothenate (4.1.1) into a 200 ml volumetric flask (take into account the moisture content giv
27、en in the suppliers certificate or dry to constant mass at 105 C) and dilute to volume with water. Store aliquots at 20 C.4.8.2 Pantothenic acid intermediate solution, = 10 g/ml. Transfer 1 ml of PA stock solution (4.8.1) into a 25 ml volumetric flask and dilute to volume with water. Store aliquots
28、at 20 C.4.8.3 Calcium pantothenate-13C6, 15N2 solution IS (Internal Standard) stock solution, = 20 g/ml. Weigh 5,0 mg of calcium pantothenate-13C6, 15N2 (4.1.2) into a 250 ml volumetric flask and dilute to volume with water. Store aliquots at 20 C.4.8.4 Solutions for the five-level standard curve. T
29、ransfer appropriate volumes of the PA intermediate solution (10 g/ml) (4.8.2) into 10 ml volumetric flasks to obtain five different concentrations of PA (0,08 g/ml, 0,16 g/ml, 0,32 g/ml, 0,64 g/ml and 1,2 g/ml). Add 500 l of the IS stock solution (20 g/ml) (4.8.3) and dilute to volume with water. Th
30、e concentration of IS in each standard solution is 1 g/ml. Store aliquots of these solutions at 20 C for no longer than one month before use.4.8.5 Ammonium acetate solution, c = 400 mmol/l, pH = 3,8 (used for sample extraction). Into a 500 ml beaker, add (30,8 0,10) g ammonium acetate. Add about 300
31、 ml water and stir to dissolve with a magnetic stirrer. Adjust to pH = 3,8 0,1, carefully adding glacial acetic acid (about 150 ml is needed). Transfer into a 1 000 ml volumetric flask and make up to volume with water. This solution is stable for one month at 4 C.1) This is an example of a suitable
32、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 same results.2 ISO 2015 All rights reservedBS ISO 20639:2015
33、ISO 20639:2015(E)5 ApparatusUsual laboratory glassware and equipment and, in particular, the following.5.1 Balances, with readability of 0,1 mg, capacity 210 g; with readability of 0,1 g, capacity 4 100 g.5.2 pH-meter, with readability of 0,01 pH unit.5.3 Homogenizer2).5.4 Stir plate with magnetic s
34、tirrers.5.5 Filters. Syringe filters, 0,22 m pore size, 33 mm internal diameter, Millex-GV PVDF (Millipore)3). Membrane disc filters, 0,45 m pore size (Millipore)3)or equivalent.5.6 UHPLC-MS/MS system, UPLC column, e.g. ACQUITY UPLC3)coupled with triple quadrupole detector equipped with electrospray
35、 ionization (ESI) source and T3 column (1,8 m, 100 mm 2,1 mm internal diameter; Waters Corp.)3)or equivalent.6 Procedure6.1 Sample preparation6.1.1 GeneralIf the product contains starch, add 50 mg -amylase to the suspensions and incubate for 15 min at 40 C to decrease viscosity and facilitate handli
36、ng. Mix liquid samples well to ensure homogeneity and continue directly to extraction. If the powder sample homogeneity is unknown, assume that it is non-homogenous and proceed with 6.1.2.6.1.2 Dry blended powder samplesFor dry blended/non-homogenous powder samples, accurately weigh approximately 25
37、,0 g (m1). Add 200,0 g (m2) water at 40 C before mixing until a homogeneous suspension is obtained. A homogenizer (5.3) can be used when necessary. Accurately weigh approximately 15,0 g (m3) aliquot of homogenized sample suspension into a 50 ml volumetric flask. Calculate the sample mass (msis the p
38、owder equivalent) using Formula (1):mmmms=132(1)wherem1is the mass of sample weighed, in g;m2is the mass of water added before mixing, in g;2) Polytron PT3000 (drive unit), Aggregate PT-DA 3012 (Kinematics, Lucerne, Switzerland) are examples of suitable products available commercially. This informat
39、ion is given for the convenience of users of this document and does not constitute an endorsement by ISO of these products. Equivalent products may be used if they can be shown to lead to the same results.3) This is an example of a suitable product available commercially. This information is given f
40、or 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. ISO 2015 All rights reserved 3BS ISO 20639:2015ISO 20639:2015(E)m3is the mass of homogenized sample suspe
41、nsion, in g.6.1.3 Wet blended powder samplesFor wet blended homogenous powder samples, accurately weigh approximately 2,0 g of sample (ms) into a 50 ml volumetric flask. Add 14 g of water at 40 C. Mix until a homogeneous suspension is obtained.6.1.4 Liquid samplesFor liquid sample samples, accuratel
42、y weigh approximately 20,0 g (ms) into a 50 ml volumetric flask.6.2 ExtractionUsing the prepared sample (6.1), add a 25 ml volume of a 0,4 mol/l ammonium acetate solution, pH = 3,8. Dilute the sample extract to volume with water. Add a stir bar and stir for 10 min. Filter a 20 ml portion through fol
43、ded paper (Grade 597). Run chromatographic analysis.6.3 Analysis6.3.1 Chromatographic analysisTransfer a 1,0 ml aliquot of the filtrate obtained in 6.2 into a 15 ml polypropylene tube containing 500 l of the IS stock solution (4.8.3). It is critical to use the same IS solution as used in the prepara
44、tion of the standard curve (4.8.4). Dilute the solution to 10 ml with water, cap and mix. Filter through a 0,22 m syringe filter (5.5). Inject into the UHPLC-MS/MS system.Examples of typical chromatograms are given in Annex A.6.3.2 UHPLC conditionsInjection volume: 2 lColumn temperature: 30 CFlow ra
45、te: 0,45 ml/minMobile phase A: 0,1 % (v/v) formic acid in waterMobile phase B: AcetonitrileThe gradient programme for the column is given in Table 1.Table 1 Gradient for columnTime minMobile phase A %Mobile phase B %0 92 82,2 80 202,4 50 504,0 50 504,1 92 87,0 92 8Direct the liquid chromatography fl
46、ow into the MS detector only between 0 min and 2 min to prevent source fouling as much as possible.4 ISO 2015 All rights reservedBS ISO 20639:2015ISO 20639:2015(E)6.3.3 MS/MS conditions Positive ESI Capillary voltage, 2,2 kV Cone voltage, 25 V Extractor voltage, 3,0 V Source temperature, 140 C Desol
47、vation temperature, 350 C Cone gas flow, 40 l/h Desolvation gas flow, 700 l/hSet the collision energy at 14 V with a dwell time for each monitored transition of 0,1 s. These values are indicative and need to be optimized for each instrument used. Monitor between 0 min and 2,1 min the transitions m/z
48、 220,2 90,1 for PA and m/z 224,2 94,1 for the isotope-labelled IS.6.3.4 IdentificationMS detection in the single-reaction monitoring mode includes simultaneous detection of molecular ions corresponding to PA and isotopically labelled PA. The selected mass transitions are m/z 220,2 90,1 and m/z 224,2
49、 94,1, respectively.7 CalculationsCalculate for each standard the peak area ratio between PA and IS. Establish a 5-point calibration curve (ranging from 0,16 ng to 2,4 ng on column) by plotting peak area ratio (y-axis) versus PA concentration (x-axis). Calculate the linear regression. It is recommended to use a weighed regression curve (1/x).Calculate the slope (S) and the intercept (I) of the calibration curve.Calculate the PA mass fraction, w, in mg/100 g, using F
