1、BSI Standards PublicationBS EN ISO 9936:2016Animal and vegetable fatsand oils Determination oftocopherol and tocotrienolcontents by high-performanceliquid chromatography (ISO9936:2016)BS EN ISO 9936:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 9936:2
2、016.It supersedes BS EN ISO 9936:2006+A1:2011 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee AW/307, Oilseeds, animal and vegetable fats and oils andtheir by-products.A list of organizations represented on this committee can beobtained on request to it
3、s secretary.This publication does not purport to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016. Published by BSI StandardsLimited 2016ISBN 978 0 580 91533 8ICS 67.200.10Compliance with a British Standard ca
4、nnot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committee on 30 April 2016.Amendments issued since publicationDate Text affectedEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 9936 April 2016 ICS 67.200.1
5、0 Supersedes EN ISO 9936:2006English 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 chrom
6、atographie en phase liquide haute performance (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 co
7、mply with the CEN/CENELEC Internal Regulations 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-CENEL
8、EC Management Centre or to any CEN member. 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
9、same status as the official versions. 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, Luxembou
10、rg, Malta, Netherlands, Norway, Poland, 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 2
11、016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 9936:2016 EBS EN ISO 9936:2016EN 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 c
12、ollaboration with Technical Committee CEN/TC 307 “Oilseeds, vegetable and animal fats and oils and their by-products - Methods 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 ide
13、ntical text or by endorsement, at the latest by October 2016, and conflicting national standards shall be withdrawn at the latest 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 hel
14、d responsible for identifying any or all such patent rights. This document supersedes EN ISO 9936:2006. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croati
15、a, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
16、 United Kingdom. Endorsement notice The text of ISO 9936:2016 has been approved by CEN as EN ISO 9936:2016 without any modification. BS EN ISO 9936:2016ISO 9936:2016(E)Foreword iv1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 15 Reagents 16 Apparatus . 27 Sampling 38 Prepa
17、ration of test sample . 39 Procedure. 39.1 Preparation of calibration solutions . 49.1.1 Stock calibration solutions 49.1.2 Standard solution 49.2 Optimization of working parameters 49.3 Preparation of test solution . 59.4 Determination . 510 Expression of results 611 Precision . 611.1 Interlaborato
18、ry test. 611.2 Repeatability . 611.3 Reproducibility . 712 Test report . 7Annex A (informative) Examples of chromatograms . 8Annex B (informative) Saponification 10Annex C (informative) Results of interlaboratory tests .12Bibliography .16 ISO 2016 All rights reserved iiiContents PageBS EN ISO 9936:2
19、016ISO 9936:2016(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 carried out through ISO technical committees. Each member body interested in a sub
20、ject 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 Commission (I
21、EC) 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 documents should be
22、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 for identifyi
23、ng 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 convenience
24、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: Foreword - Suppl
25、ementary 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 Amendment ISO 9
26、936:2006/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.iv ISO 2016 All rights reservedBS EN ISO 9936:2016INTERNATIONAL STANDARD ISO 9936:2016(E)Animal and vegetable fats and oils Determination o
27、f tocopherol and tocotrienol contents by high-performance liquid chromatography1 ScopeThis International Standard specifies a method for the determination of the contents of free -, -, -, and -tocopherols and tocotrienols (referred to jointly as tocols) in animal and vegetable fats and oils (referre
28、d to hereinafter as fats) by high-performance liquid chromatography (HPLC).For products containing tocopherol or tocotrienol esters, it is necessary to carry out a preliminary saponification.Milk and milk products (or fat coming from milk and milk products) are excluded from the Scope of this Intern
29、ational Standard.NOTE A suitable method involving a cold saponification procedure is described in Annex B for information only.2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated referenc
30、es, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 661, Animal and vegetable fats and oils Preparation of test sample3 Terms and definitionsFor the purposes of this document, the following terms and definit
31、ions apply.3.1tocol contentmass fraction of the individual tocols, determined using the method specified in this International StandardNote 1 to entry: The content is expressed in milligrams per kilogram as a whole number.4 PrincipleA test portion is dissolved in n-heptane and the individual tocols
32、are separated by high-performance liquid chromatography (HPLC). The content of each tocol is calculated using calibration factors determined from calibration solutions.5 ReagentsUse only reagents of HPLC grade or equivalent.5.1 -, -, - and -tocopherol and tocotrienol standards.If tocopherol standard
33、s are not available, a blend of wheat germ and soya bean oil may be used to identify -, -, - and -tocopherols. ISO 2016 All rights reserved 1BS EN ISO 9936:2016ISO 9936:2016(E)If tocotrienol standards are not available, palm oil may be used to identify - and -tocotrienols. The chromatograms obtained
34、 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 -tocopherol and tocotrienol standards can be obtained from Merck1); -tocopherol can be obtained from various suppliers. Toc
35、otrienol 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 %. Thus, it is important to determine the concentration of prepared calibration solutions by UV spectrometry (see 9.1.1).5.2 T
36、etrahydrofuran, 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 that has been proven to reach a chromatographic resolution of peaks as good as the one presented in Table 2 (relative re
37、tention 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 -tocotrienol can be achieved by using a mixture of 5 % volume fraction t-butyl methyl ether + 95 % volume fraction n-heptan
38、e 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 (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
39、 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 following.6.1 HPLC system, consisting of a high-pressure pump, a sample injection device, column thermostat adjusted to 25
40、 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 be used if a fluorescence detector is not available but it is not recommended. However, if a UV detector is used, the wav
41、elength 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 mm 4,6 mm, packed with microparticulate silica having a mean particle size of about 5 m.1) Merck Tocopherol set 613424 i
42、s 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). Merck Tocotrienol set 613432 is available from Calbiochem also. It contains one 50 mg vial each of -tocotrienol, -tocotri
43、enol, -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
44、 same results.2) Tocotrienols are available from Sigma Aldrich () and from Chromadex () with purities between 65 % and 98 %. 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
45、 they can be shown to lead to the same results.2 ISO 2016 All rights reservedBS EN ISO 9936:2016ISO 9936:2016(E)NOTE 1 Suitable diol silica column packing material available commercially is 5 m LiChrospher 100 Diol; suitable silica column packing materials available commercially are 5 m LiChrosob SI
46、 60 and Kromasil 1003). When -tocotrienol is expected in the sample, the diol silica column is preferred as -tocopherol and -tocotrienol are co-eluted when using the silica column.NOTE 2 The length and the diameter of the column can be varied according to the HPLC technique used.NOTE 3 Both types of
47、 columns have been used for the evaluation of the precision data (Annex C).6.3 UV spectrometer, capable of absolute measurement of absorbance at precisely defined wavelengths, with a 10-mm path length cell.6.4 Rotary evaporator.6.5 Graduated cylinder, of 1 000 ml capacity.6.6 Volumetric pipettes, of
48、 5 ml, 10 ml and 20 ml capacities.6.7 Volumetric flasks, 50 ml and 25 ml capacities.6.8 Ultrasonic bath.7 SamplingA representative sample should be sent to the laboratory. It is important that the sample has not been damaged or changed during transport or storage.Sampling is not part of the method s
49、pecified in this International Standard. A recommended sampling method is given in ISO 5555.8 Preparation of test sampleIn the case of liquid laboratory samples, prepare the test sample by homogenization as described in ISO 661, except that filtration should be avoided.In the case of solid samples, transfer a representative portion (i.e. not less than 10 % by mass of the laboratory sample) to a glass beaker and carefully homogenize by melting, with
