1、BS ISO23065:2009ICS 67.100.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDMilk fat from enricheddairy products Determinationof omega-3 andomega-6 fatty acidcontent by gas-liquidchromatographyThis British Standardwas published under theauthority of the Standar
2、dsPolicy and StrategyCommittee on 31 October2009 BSI 2009ISBN 978 0 580 55197 0Amendments/corrigenda issued since publicationDate CommentsBS ISO 23065:2009National forewordThis British Standard is the UK implementation of ISO 23065:2009.The UK participation in its preparation was entrusted to Techni
3、calCommittee AW/5, Chemical analysis of milk and milk products.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Co
4、mpliance with a British Standard cannot confer immunityfrom legal obligations.BS ISO 23065:2009Reference numbersISO 23065:2009(E)IDF 211:2009(E)ISO and IDF 2009INTERNATIONAL STANDARD ISO23065IDF211First edition2009-03-01Milk fat from enriched dairy products Determination of omega3 and omega6 fatty a
5、cid content by gas-liquid chromatography Matire grasse laitire de produits laitiers enrichis Dtermination de la teneur en acides gras omga3 et omga6 par chromatographie gaz-liquide BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) PDF disclaimer This PDF file may contain embedded typefaces. In acco
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9、OCUMENT ISO and IDF 2009 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO or IDF at the respective addr
10、ess below. ISO copyright office International Dairy Federation Case postale 56 CH-1211 Geneva 20 Diamant Building Boulevard Auguste Reyers 80 B-1030 Brussels Tel. + 41 22 749 01 11 Tel. + 32 2 733 98 88 Fax + 41 22 749 09 47 Fax + 32 2 733 04 13 E-mail copyrightiso.org E-mail infofil-idf.org Web www
11、.iso.org Web www.fil-idf.org Published in Switzerland ii ISO and IDF 2009 All rights reservedBS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) ISO and IDF 2009 All rights reserved iiiContents Page Foreword iv Foreword. v 1 Scope 1 2 Normative references 1 3 Terms and definitions .1 4 Principle2 5 R
12、eagents.2 6 Apparatus .3 7 Sampling.5 8 Preparation of test sample5 9 Procedure .5 9.1 Calibration solution for the determination of LA, EPA and DHA response factor5 9.2 Test portion 5 9.3 Qualitative determination5 9.4 Quantitative determination .6 9.5 Calculation and expression of results.6 9.6 Ex
13、pression of results 7 10 Precision.7 10.1 Repeatability.7 10.2 Reproducibility.7 11 Test report 7 Annex A (informative) Examples of gas-liquid chromatographic analysis.8 Annex B (informative) Interlaboratory trial .11 Bibliography 12 BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) iv ISO and IDF
14、2009 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide 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
15、 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. ISO collaborates closely with the International Electrotechnical Commiss
16、ion (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical
17、committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall
18、not be held responsible for identifying any or all such patent rights. ISO 23065IDF 211 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products. It is being published jointly by ISO and IDF. BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) ISO and ID
19、F 2009 All rights reserved vForeword IDF (the International Dairy Federation) is a non-profit organization representing the dairy sector worldwide. IDF membership comprises National Committees in every member country as well as regional dairy associations having signed a formal agreement on cooperat
20、ion with IDF. All members of IDF have the right to be represented at the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action
21、Teams and Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of IDF National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subje
22、ct of patent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 23065IDF 211 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF). It is being published jointly
23、by IDF and ISO. All work was carried out by the Joint ISO-IDF Action Team on Fat, of the Standing Committee on Main components in milk, under the aegis of its project leaders: Mrs. G. Contarini (IT). BS ISO 23065:2009BS ISO 23065:2009INTERNATIONAL STANDARD ISO 23065:2009(E)IDF 211:2009(E) ISO and ID
24、F 2009 All rights reserved 1Milk fat from enriched dairy products Determination of omega3 and omega6 fatty acid content by gas-liquid chromatography 1 Scope This International Standard specifies a method for the determination of the omega3 ( 3) and omega6 ( 6) fatty acid content in anhydrous milk fa
25、t extracted from dairy products supplemented or naturally enriched with these constituents. The specified procedure allows the evaluation of the most important 3 and 6 fatty acids. NOTE The notations “omega-3”, “-3” and “3” are erroneous, but in common use, they are equivalent to “ 3”. The same is t
26、rue for “omega-6”, “-6”, and “6”, which are equivalent to “ 6”. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced documen
27、t (including any amendments) applies. ISO 14156IDF 172, Milk and milk products Extraction methods for lipids and liposoluble compounds ISO 15884IDF 182, Milk fat Preparation of fatty acid methyl esters 3 Terms and definitions For the purposes of this International Standard, the following terms and d
28、efinitions apply. 3.1 3 fatty acid omega3 fatty acid omega-3 fatty acid (deprecated) -3 fatty acid (deprecated) 3 fatty acid (deprecated) polyunsaturated fatty acid having the first double bond three carbons from the terminal methyl group 3.2 6 fatty acid omega6 fatty acid omega-6 fatty acid (deprec
29、ated) -6 fatty acid (deprecated) 6 fatty acid (deprecated) polyunsaturated fatty acid having the first double bond six carbons from the terminal methyl group BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) 2 ISO and IDF 2009 All rights reserved3.3 3 and 6 fatty acid content mass fraction of subst
30、ances determined by the procedure specified in this International Standard NOTE The 3 and 6 fatty acid content is expressed as a mass fraction in milligrams per 100 g of fat of the fatty acids listed in Table 1. Table 1 Constituents of 3 and 6 fatty acid content Chain length and double bond designat
31、ion Systematic name Trivial name and/or abbreviated term C18:2 6 (9Z,12Z)-octadeca-9,12-dienoic acid linoleic acid, LA C18:3 3 (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid linolenic acid, LNA C18:4 3 (6Z,9Z,12Z,15Z)-octadeca-6,9,12,15-tetraenoic acid stearidonic acid C20:5 3 (5Z,8Z,11Z,14Z,17Z)-eicos
32、a-5,8,11,14,17-pentaenoic acid EPA C22:5 3 (7Z,10Z,13Z,16Z,19Z)-docosa-7,10,13,16,19-pentaenoic acid DPA C22:6 3 (4Z,7Z,10Z,13Z,16Z,19Z)-docosa-4,7,10,13,16,19-hexaenoic acid DHA 4 Principle An internal standard is added to anhydrous milk fat. Fatty acid methyl esters (FAMEs) are prepared by transes
33、terification. The FAMEs are separated and determined by capillary gas chromatography. Individual 3 and 6 fatty acids are quantified by reference to the internal standard. 5 Reagents Use only reagents of recognized analytical grade, and only distilled or demineralized water or water of equivalent pur
34、ity. 5.1 n-Hexane CH3(CH2)4CH3. 5.2 C23:0 (tricosanoic) FAME, of purity 99 % mass fraction. 5.3 C23:0 FAME standard solution. Accurately weigh approximately 25 mg of C23:0 (5.2) in a 25 ml one-mark volumetric flask (6.3). Dilute to the mark with n-hexane (5.1) and mix. The C23:0 standard solution ma
35、y be stored in a refrigerator for one month. 5.4 C20:5 3 (EPA) FAME, of purity 99 % mass fraction. 5.5 C20:5 3 FAME standard solution. Accurately weigh approximately 10 mg of C20:5 3 (5.4) in a 10 ml one-mark volumetric flask (6.3). Dilute to the mark with n-hexane (5.1) and mix. 5.6 C22:6 3 (DHA) F
36、AME, of purity 99 % mass fraction. 5.7 C22:6 3 FAME standard solution. Accurately weigh approximately 10 mg of C22:6 3 (5.6) in a 10 ml one-mark volumetric flask (6.3). Dilute to the mark with n-hexane (5.1) and mix. BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) ISO and IDF 2009 All rights rese
37、rved 35.8 C18:2 6 (LA) FAME, of purity 99 % mass fraction. 5.9 C18:2 6 FAME standard solution. Accurately weigh approximately 10 mg of C18:2 6 (5.8) in a 10 ml one-mark volumetric flask (6.3). Dilute to the mark with n-hexane (5.1) and mix. 5.10 Reference mixture, consisting of 3 and 6 fatty acid me
38、thyl esters, for qualitative evaluation, i.e. identification of retention times. The reference mixture should contain the FAMEs of the acids listed in Table 1 together with the internal standard C23:0 methyl ester (5.2). It may be obtained by mixing 1 ml of the four standard solutions (5.3, 5.5, 5.7
39、, and 5.9) with 1 ml of the other three solutions prepared by weighing linolenic, stearidonic and DPA FAMEs (purity at least 80 % mass fraction) at the same concentration in n-hexane (1 mg/ml). Due to the high cost of some of these 3 FAMEs, commercially available mixtures (including other FAMEs) may
40、 replace the reference mixture. If a commercial mixture of polyunsaturated FAMEs is used, take into account the concentration indicated and, if necessary, adjust with n-hexane. If not already present, add a suitable amount of C23:0 FAME standard solution (5.3). 6 Apparatus Usual laboratory equipment
41、 and, in particular, the following. 6.1 Analytical balance, capable of being read to the nearest 0,1 mg. 6.2 Drying oven, capable of being maintained at 60 C 2 C. 6.3 One-mark volumetric flasks, of capacities 10 ml, 25 ml and 50 ml, ISO 10424class A. 6.4 One-mark pipettes, of capacities 1 ml and 5 m
42、l, ISO 6481class A. 6.5 Graduated pipette, of capacity 5 ml, ISO 8353class A. 6.6 Test tube, of capacity 10 ml, fitted with polytetrafluoroethylene-lined screw cap. 6.7 Gas-liquid chromatograph, equipped with flame ionization detector and capillary split injection system or on-column. 6.7.1 Carrier
43、gas, hydrogen or helium of purity grade at least 99,999 % mass fraction. 6.7.2 Other gases, with no detectable trace of organic impurities (hydrocarbon content less than 1 mg/kg), nitrogen and hydrogen, purity at least 99,995 % mass fraction, and synthetic air. 6.7.3 Capillary column, with a station
44、ary phase which has been successfully employed to perform separation of FAMEs. The use of polyethylene glycol phase capillary columns is suggested, of length 25 m, 30 m or 60 m, of internal diameter 0,25 mm or 0,32 mm and of film thickness 0,12 m or 0,25 m. These columns elute the FAMEs primarily by
45、 carbon chain length and secondarily by the number of double bonds. It is possible to use more polar phases, such as cyanosilicone stationary phases, but, in this case, particular care should be taken in peak identification; moreover with increasing polarity, a good separation between the C23:0 FAME
46、, adopted as internal standard, and the C20:5 3 FAME is increasingly difficult to obtain. BS ISO 23065:2009ISO 23065:2009(E) IDF 211:2009(E) 4 ISO and IDF 2009 All rights reserved6.7.4 Gas chromatographic conditions. The oven temperature and the carrier gas flow depend on the column selected and on
47、the carrier gas adopted. In any case, the selected conditions shall produce separation between C18:4 3 and C18:2 conjugated linoleic acid (CLA) and between DPA and DHA (see Figures A.1 and A.2). See Examples 1 and 2 for conditions. EXAMPLE 1 Using helium as carrier gas and isothermal oven conditions
48、, applicable conditions for correct separation of 3 and 6 FAMEs are: Carrier gas: Helium at a flow rate of 1,8 ml/min Column head pressure: 120 kPa Column: Fused silica capillary Length 30 m Internal diameter 0,32 mm Film thickness 0,25 m Stationary phase: Polyethylene glycol Column temperature: 200
49、 C Injector and detector temperature: 260 C Split ratio: 100:1 Volume of sample injected: 1 l A gas-liquid chromatogram obtained under these conditions is shown in Figure A.1. EXAMPLE 2 Using hydrogen as carrier gas and programmed oven conditions, applicable conditions for correct separation of 3 and 6 FAMEs are: Carrier gas: Hydrogen at a constant
