1、BSI Standards PublicationBS EN ISO 12228-2:2014Determination of individualand total sterols contents Gas chromatographic methodPart 2: Olive and olive pomace oilsBS EN ISO 12228-2:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 12228-2:2014. Together wi
2、th BS EN ISO 12228-1:2014 it supersedes BS EN ISO 12228:1999, which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and their by-products.A list of organizations represented on this committee can be obtain
3、ed on request to its secretary.This publication does not purport to include all the necessary 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 77277 1 ICS 67.200.10 Compliance wit
4、h a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 January 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUR
5、OPISCHE NORM EN ISO 12228-2 October 2014 ICS 67.200.10 Supersedes EN ISO 12228:1999English Version Determination of individual and total sterols contents - Gas chromatographic method - Part 2: Olive and olive pomace oils (ISO 12228-2:2014) Dtermination de la teneur en strols individuels et totaux -M
6、thode par chromatographie en phase gazeuse - Partie 2: Huile dolive et huile de grignons dolive (ISO 12228-2:2014)Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches Verfahren - Teil 2: Oliven- und Oliventresterle (ISO 12228-2:2014) This European Standard was approved by CEN
7、on 6 September 2014. CEN members are bound to comply 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
8、 may be obtained on application to the CEN-CENELEC 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 notifi
9、ed to the CEN-CENELEC Management Centre has the 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, Icel
10、and, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Manag
11、ement Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 12228-2:2014 EBS EN ISO 12228-2:2014EN ISO 12228-2:2014 (E) 3 Foreword This document (EN ISO 12228-2:2014) has been prepared b
12、y 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 of sampling and analysis” the secretariat of which is held by AFNOR. This European Standard shall be given the status of
13、a national standard, either by publication of an identical text or by endorsement, at the latest by April 2015, and conflicting national standards shall be withdrawn at the latest by April 2015. Attention is drawn to the possibility that some of the elements of this document may be the subject of pa
14、tent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN ISO 12228:1999. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this Euro
15、pean Standard: 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, Poland, Portugal, Romania, Slovakia, Slo
16、venia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 12228-2:2014 has been approved by CEN as EN ISO 12228-2:2014 without any modification. BS EN ISO 12228-2:2014ISO 12228-2:2014(E) ISO 2014 All rights reserved iiiContents PageForeword iv1 Scope . 12 N
17、ormative references 13 Terms and definitions . 14 Principle 25 Reagents 26 Apparatus . 37 Sample . 47.1 Sampling . 47.2 Preparation of the test sample 48 Procedure. 48.1 Test portion 48.2 Preparation of unsaponifiable matter. 48.3 Separation of the sterol and triterpene dialcohols (erythrodiol, uvao
18、l) fractions by TLC 58.4 Preparation of the trimethylsilyl ethers 68.5 Gas chromatographic analysis . 69 Expression of results 79.1 Quantitative evaluation . 79.2 Determination of the total sterol content 89.3 Composition of sterols 89.4 Composition of triterpene dialcohols 810 Precision . 910.1 Int
19、erlaboratory test. 910.2 Repeatability limit, r . 910.3 Reproducibility limit, R . 911 Test report . 9Annex A (informative) Figures .10Annex B (informative) Interlaborative trial .13Bibliography .16BS EN ISO 12228-2:2014ISO 12228-2:2014(E)ForewordISO (the International Organization for Standardizati
20、on) 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 subject for which a technical committee has been established has the right to be re
21、presented 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 (IEC) on all matters of electrotechnical standardization.The procedures used to de
22、velop 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 noted. This document was drafted in accordance with the editorial rules of the I
23、SO/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 identifying any or all such patent rights. Details of any patent rights identified during
24、 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 of users and does not constitute an endorsement.For an explanation on the meanin
25、g 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 - Supplementary informationThe committee responsible for this document is ISO/TC 34, Fo
26、od products, Subcommittee SC 11, Animal and vegetable fats and oils.This first edition of ISO 12228-2 cancels and replaces ISO 12228:1999, which has been technically revised.ISO 12228 consists of the following parts, under the general title Determination of individual and total sterols contents Gas
27、chromatographic method: Part 1: Animal and vegetable fats and oils Part 2: Olive oils and olive pomace oilsiv ISO 2014 All rights reservedBS EN ISO 12228-2:2014INTERNATIONAL STANDARD ISO 12228-2:2014(E)Determination of individual and total sterols contents Gas chromatographic method Part 2: Olive oi
28、ls and olive pomace oils1 ScopeThis part of ISO 12228 specifies a procedure for the gas chromatographic determination of the contents and composition of sterols and triterpene dialcohols in olive and olive pomace oils. For the determination of the contents and composition of sterols in all other ani
29、mal and vegetable fats and oils, ISO 12228-1 is to be used.NOTE This part of ISO 12228 is technically identical to IOC Standard COI/T.20/Doc. No. 30 (November 2011).2 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable f
30、or 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 vegetable fats and oils Preparation of test sample3 Terms and definitionsFor the purposes of this doc
31、ument, the following terms and definitions apply.3.1composition of sterolscomposition of individual sterols in the sample, beginning with cholesterol and ending with 7-avenasterol (see Table 1) under the conditions specified in this part of ISO 12228Note 1 to entry: The composition is expressed as a
32、 percentage of all peak areas, normalized to 100 %.3.2total sterol contentmass fraction of the sum of all individual sterols, as determined in accordance with the method specified in this part of ISO 12228, beginning with cholesterol and ending with 7-avenasterol (see Table 1), divided by the mass o
33、f the test portionNote 1 to entry: The content is expressed in milligrams per kilogram.3.3composition of triterpene dialcoholscomposition of erythrodiol and uvaol in the sample under the conditions specified in this part of ISO 12228Note 1 to entry: The composition is expressed as a percentage of al
34、l peak areas, beginning with cholesterol and ending with uvaol (see Table 1) under the conditions specified in this part of ISO 12228, normalized to 100 %. ISO 2014 All rights reserved 1BS EN ISO 12228-2:2014ISO 12228-2:2014(E)4 PrincipleA test portion is saponified by boiling under reflux with an e
35、thanolic potassium hydroxide solution. The unsaponifiable matter is extracted with diethyl ether. The sterol and triterpene dialcohol fractions are separated from the unsaponifiable matter by thin-layer chromatography on a basic silica gel plate. The qualitative and quantitative compositions of the
36、sterol and triterpene dialcohol fractions are determined by gas chromatography of the trimethylsilyl ethers using cholestanol as internal standard.5 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational and personal safety
37、 measures shall be followed.Use only reagents of recognized analytical grade, unless otherwise stated, and water complying with grade 3 of ISO 3696.15.1 Potassium hydroxide, minimum mass fraction w = 85 g/100 g.5.2 Potassium hydroxide, ethanolic solution, amount concentration, c, approximately 2 mol
38、/l.While cooling, dissolve 130 g of potassium hydroxide (5.1) in 200 ml of distilled water and then make up to 1 l with ethanol (5.9). Keep the solution in well-stoppered dark glass bottles and store for a maximum of 2 d.5.3 Potassium hydroxide, ethanolic solution, amount concentration, c, approxima
39、tely 0,2 mol/l.Dissolve 13 g of potassium hydroxide (5.1) in 20 ml of distilled water and make up to 1 l with ethanol (5.9).5.4 Diethyl ether, for chromatography.WARNING Diethyl ether is highly flammable and can form explosive peroxides. Explosive limits in air are 1,7 % to 48 % (volume fraction). T
40、ake special precautions when using it.5.5 Anhydrous sodium sulfate.5.6 Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions 20 cm 20 cm, thickness of layer 0,25 mm, without fluorescence indicator.5.7 Acetone, for chromatography.5.8 n-Hexane, for chromatography.5.9 Et
41、hanol 96 %, minimum volume fraction = 95 %.5.10 Ethyl acetate.5.11 Reference solution for thin-layer chromatography, cholesterol or mixture of phytosterols, and erythrodiol solution in ethyl acetate (5.10), mass concentration, = 5 %.5.12 2,7-dichlorofluorescein, ethanolic solution, mass concentratio
42、n, = 0,2 %.Make slightly basic by adding a few drops of 2 mol/l alcoholic potassium hydroxide solution (5.2). Store for a maximum of 1 year.2 ISO 2014 All rights reservedBS EN ISO 12228-2:2014ISO 12228-2:2014(E)5.13 -cholestanol internal standard solution, mass concentration, = 0,2 g/100 ml, in ethy
43、l acetate (5.10).5.14 Phenolphthalein solution, mass concentration, = 10 g/l, in ethanol (5.9).5.15 Carrier gas for gas chromatography, helium or preferably hydrogen.5.16 Auxiliary gases for gas chromatography, hydrogen, helium, nitrogen, and air.5.17 Developing solvent, mixture of n-hexane and diet
44、hyl ether, volume concentrations are: (n-hexane) = 65 ml/100 ml, (diethyl ether) = 35 ml/100 ml.5.18 Hexamethyldisilazane.5.19 Trimethylchlorosilane.5.20 Silylation reagent, mixture of pyridine, hexamethyldisilazane, and trimethylchlorosilane.Volume concentration (pyridine) = 9 ml/13 ml, (hexamethyl
45、 disilazane) = 3 ml/13 ml, (trimethylchlorosilane) = 1 ml/13 ml. Prepare the mixture fresh daily.NOTE Other silylation reagents can be used, e. g., mixture of N,O-bis-trimethylsilyl-trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS), (BSTFA) = 99 ml/100 ml and (TMSC) = 1 ml/100ml.6 Apparatu
46、sUsual laboratory apparatus and, in particular, the following.6.1 Round-bottomed flasks, of 250 ml, with ground neck.6.2 Reflux condenser, with ground glass joint to fit the flask (6.1).6.3 Separating funnel, of 500 ml capacity.6.4 Developing tank, made of glass, with a ground glass lid, suitable fo
47、r use with plates of dimensions 20 cm 20 cm.6.5 Ultraviolet lamp, wavelength of 366 nm or 254 nm.6.6 Microsyringe, to deliver 100 l, 500 l, and 1000 l.6.7 Cylindrical filter funnel, with sintered-glass filter (G3, porosity 15 m to 40 m), diameter approximately 2 cm, depth 5 cm, suitable for filtrati
48、on under vacuum with male ground glass joint.6.8 Conical flask, for operation under a vacuum, 50 ml with ground glass female joint to fit to the filter funnel (6.7).6.9 Test tube, 10 ml with a tapering bottom and a sealing glass stopper.6.10 Gas chromatograph, for capillary columns, with split injec
49、tor, consisting of: column oven, capable of maintaining the temperature with an accuracy of 1C; ISO 2014 All rights reserved 3BS EN ISO 12228-2:2014ISO 12228-2:2014(E) temperature-controlled injection unit, with persilanised glass vaporising element and split system; flame ionization detector (FID); data acquisition system.6.11 Capillary column, made of fused silica, length 20 m to 30 m, internal diameter 0,25 mm to 0,32 mm, coated with 5 % Diphenyl, 95 % Dimethyl polysiloxane (SE-52 or SE-54 or equivalent st
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