DIN EN ISO 9936-2016 Animal and vegetable fats and oils - Determination of tocopherol and tocotrienol contents by high-performance liquid chromatography (ISO 9936 2016) German vers.pdf

1、November 2016 English price group 12No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 67.200.10!%q+“2587808www.din.de

2、DIN EN ISO 9936Animal and vegetable fats and oils Determination of tocopherol and tocotrienol contents by highperformanceliquid chromatography (ISO 9936:2016);English version EN ISO 9936:2016,English translation of DIN EN ISO 9936:2016-11Tierische und pflanzliche Fette und le Bestimmung des Tocopher

3、ol und TocotrienolGehaltes mittels Hochleistungsflssigchromatographie (ISO 9936:2016);Englische Fassung EN ISO 9936:2016,Englische bersetzung von DIN EN ISO 9936:2016-11Corps gras dorigines animale et vgtale Dtermination des teneurs en tocophrols et en tocotrinols par chromatographie en phase liquid

4、e haute performance (ISO 9936:2016);Version anglaise EN ISO 9936:2016,Traduction anglaise de DIN EN ISO 9936:2016-11SupersedesDIN EN ISO 9936:201111www.beuth.deDocument comprises tt pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritati

5、ve.11.16DIN EN ISO 9936:2016-11 2 A comma is used as the decimal marker. National foreword This document (EN ISO 9936:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with Technical Committee CEN/TC 307 “Oil seeds, vegetable and a

6、nimal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products) and the

7、Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Working Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standards corresponding to the International Standards referred to in this docu

8、ment are as follows: ISO 661 DIN EN ISO 661 ISO 3696 DIN ISO 3696 ISO 5555 DIN EN ISO 5555 ISO 5725-1 DIN ISO 5725-1 ISO 5725-2 DIN ISO 5725-2 Amendments This standard differs from DIN EN ISO 9936:2011-11 as follows: a) milk and milk products (or fat coming from milk and milk products) are now exclu

9、ded from the scope of the standard; b) the standard has been editorially revised and brought in line with the current rules of presentation. Previous editions DIN EN ISO 9936: 2006-07, 2011-11 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation

10、 of test sample DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 3696, Water for analytical laboratory use Specification and test methods DIN ISO 5725-1, Accuracy (trueness and precision) of measurement methods and results Part 1: General principles and definitions DIN ISO 5725-2

11、, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 9936 April 2016 ICS 67.200.10 Supersedes EN ISO 9936:2006En

12、glish Version Animal and vegetable fats and oils Determination of tocopherol and tocotrienol contents by high-performance liquid chromatography (ISO 9936:2016) Corps gras dorigines animale et vgtale Dtermination des teneurs en tocophrols et en tocotrinols par chromatographie en phase liquide haute p

13、erformance (ISO 9936:2016) Tierische und pflanzliche Fette und le Bestimmung des Tocopherol- und Tocotrienol-Gehaltes mittels Hochleistungsflssigchromatographie (ISO 9936:2016) This European Standard was approved by CEN on 4 March 2016. CEN members are bound to comply with the CEN/CENELEC Internal R

14、egulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN m

15、ember. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions

16、. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Pola

17、nd, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation i

18、n any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 9936:2016 EEN ISO 9936:2016 (E) 2 Contents Page European foreword . 3 Foreword 4 1 Scope 5 2 Normative references 5 3 Terms and definitions . 5 4 Principle . 5 5 Reagents . 5 6 Apparatus . 6 7 Sampling . 7 8 Pre

19、paration of test sample 7 9 Procedure. 7 9.1 Preparation of calibration solutions 8 9.1.1 Stock calibration solutions 8 9.1.2 Standard solution 8 9.2 Optimization of working parameters 8 9.3 Preparation of test solution 9 9.4 Determination 9 10 Expression of results . 10 11 Precision 10 11.1 Interla

20、boratory test . 10 11.2 Repeatability 10 11.3 Reproducibility . 11 12 Test report 11 Annex A (informative) Examples of chromatograms . 12 Annex B (informative) Saponification . 14 B.1 General . 14 B.2 Reagents 14 B.3 Procedure 14 Annex C (informative) Results of interlaboratory tests 16 Bibliography

21、 . 20 DIN EN ISO 9936:2016-11EN ISO 9936:2016 (E) 3 European foreword This document (EN ISO 9936:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods

22、 of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by October 2016, and conflicting national standards shall be withdrawn at the lat

23、est by October 2016. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 9936:2006. According to the CEN-C

24、ENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary,

25、 Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 9936:2016 has been approved by CEN as EN ISO 9936:2016 without any modific

26、ation. DIN EN ISO 9936:2016-11 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 carried out through ISO technical committees. Each member body interes

27、ted 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. ISO collaborates closely with the International Electrotechnical C

28、ommission (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 different approval criteria needed for the different types of ISO document

29、s should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, 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 f

30、or 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 (see www.iso.org/patents).Any trade name used in this document is information given for the

31、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 Technical Barriers to Trade (TBT) see the following URL: Fore

32、word - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 11, Animal and vegetable fats and oils.This third edition cancels and replaces the second edition (ISO 9936:2006), which has been technically revised. It also incorporates the Ame

33、ndment ISO 9936:2006/Amd.1:2011 and the Technical Corrigendum ISO 9936:2006/Cor.1:2008. A non-applicability statement for milk and milk products has been added to the Scope.4EN ISO 9936:2016 (E) DIN EN ISO 9936:2016-11 1 ScopeThis International Standard specifies a method for the determination of th

34、e contents of free -, -, -, and -tocopherols and tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils (referred to hereinafter as fats) by high-performance liquid chromatography (HPLC).For products containing tocopherol or tocotrienol esters, it is necessary to carry ou

35、t a preliminary saponification.Milk and milk products (or fat coming from milk and milk products) are excluded from the Scope of this International Standard.NOTE A suitable method involving a cold saponification procedure is described in Annex B for information only.2 Normative referencesThe followi

36、ng documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 661, Animal and

37、 vegetable fats and oils Preparation of test sample3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1tocol contentmass fraction of the individual tocols, determined using the method specified in this International StandardNote 1 to entry: The cont

38、ent is expressed in milligrams per kilogram as a whole number.4 PrincipleA test portion is dissolved in n-heptane and the individual tocols are separated by high-performance liquid chromatography (HPLC). The content of each tocol is calculated using calibration factors determined from calibration so

39、lutions.5 ReagentsUse only reagents of HPLC grade or equivalent.5.1 -, -, - and -tocopherol and tocotrienol standards.If tocopherol standards are not available, a blend of wheat germ and soya bean oil may be used to identify -, -, - and -tocopherols.5 EN ISO 9936:2016 (E) DIN EN ISO 9936:2016-11 If

40、tocotrienol standards are not available, palm oil may be used to identify - and -tocotrienols. The chromatograms obtained can be used to assist peak identification in test sample chromatograms, in which case the calibration factors for the corresponding tocopherols should be used.NOTE -, -, - and -t

41、ocopherol and tocotrienol standards can be obtained from Merck1); -tocopherol can be obtained from various suppliers. Tocotrienol standards are available from Sigma Aldrich2). It has been reported that the purity of some commercially available tocopherol standards may vary between 85 % and 100 %. Th

42、us, it is important to determine the concentration of prepared calibration solutions by UV spectrometry (see 9.1.1).5.2 Tetrahydrofuran, filtered through an HPLC nylon filter (0,45 m).5.3 n-Heptane, filtered through an HPLC nylon filter (0,45 m).5.4 HPLC mobile phase.Any suitable mixture of solvents

43、 that has been proven to reach a chromatographic resolution of peaks as good as the one presented in Table 2 (relative retention time of tocopherols and tocotrienols) and in Annex A (chromatograms of a mixture of vegetable oils), should be used (see Table C.3).A good separation of -tocopherol and -t

44、ocotrienol can be achieved by using a mixture of 5 % volume fraction t-butyl methyl ether + 95 % volume fraction n-heptane and a diol-column. The preparation of a suitable mobile phase, 3,85 % (volume fraction) tetrahydrofuran solution in n-heptane, is as follows. Using a 1 000 ml graduated cylinder

45、 (6.5), introduce 1 000 ml of n-heptane (5.3) in a 2 litre bottle. Add twice 20 ml of tetrahydrofuran (5.2) using a 20 ml volumetric pipette (6.6). Homogenize the mobile phase by means of an ultrasonic bath (6.8) for 15 min.5.5 Methanol.6 ApparatusUsual laboratory apparatus and, in particular, the f

46、ollowing.6.1 HPLC system, consisting of a high-pressure pump, a sample injection device, column thermostat adjusted to 25 C (optional), a fluorescence detector with the excitation wavelength set at 295 nm and emission wavelength at 330 nm, and a recording integrator.An ultraviolet (UV) detector may

47、be used if a fluorescence detector is not available but it is not recommended. However, if a UV detector is used, the wavelength should be set at 292 nm.6.2 HPLC analytical column, two types are possible: 250 mm 4 mm, packed with microparticulate diol having a mean particle size of about 5 m, or 250

48、 mm 4,6 mm, packed with microparticulate silica having a mean particle size of about 5 m.1) Merck Tocopherol set 613424 is available from Calbiochem (). It contains one 50 mg vial each of dl-tocopherol, d-tocopherol, d-tocopherol, and d-tocopherol with a purity of 95 % by HPLC (for each component).

49、Merck Tocotrienol set 613432 is available from Calbiochem also. It contains one 50 mg vial each of -tocotrienol, -tocotrienol, -tocotrienol, and -tocotrienol with a purity of 95 % by HPLC (75 % for -tocotrienol). This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of the products named. Equivalent products may be used if they can be shown to lead to the same results.2) Tocotrienols are available