DIN EN ISO 12228-2-2015 Determination of individual and total sterols contents - Gas chromatographic method - Part 2 Olive and olive pomace oils (ISO 12228-2 2014) German version E.pdf

1、February 2015 Translation by DIN-Sprachendienst.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).I

2、CS 67.200.10!%B)“2293106www.din.deDDIN EN ISO 12228-2Determination of individual and total sterols contents Gas chromatographic method Part 2: Olive and olive pomace oils (ISO 12228-2:2014);English version EN ISO 12228-2:2014,English translation of DIN EN ISO 12228-2:2015-02Bestimmung der individuel

3、len und der Gesamtsterine Gaschromatographisches Verfahren Teil 2: Oliven- und Oliventresterle (ISO 12228-2:2014);Englische Fassung EN ISO 12228-2:2014,Englische bersetzung von DIN EN ISO 12228-2:2015-02Dtermination de la teneur en strols individuels et totaux Mthode par chromatographie en phase gaz

4、euse Partie 2: Huile dolive et huile de grignons dolive (ISO 12228-2:2014);Version anglaise EN ISO 12228-2:2014,Traduction anglaise de DIN EN ISO 12228-2:2015-02Together withDIN EN ISO12228-1:2014-10,supersedesDIN EN ISO 12228:1999-05www.beuth.deDocument comprises 23 pagesIn case of doubt, the Germa

5、n-language original shall be considered authoritative.01.15 DIN EN ISO 12228-2:2015-02 2 A comma is used as the decimal marker. National foreword This document (EN ISO 12228-2:2014) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with

6、Technical Committee CEN/TC 307 “Oil seeds, vegetable and animal fats and oils and their by-products Methods of sampling and analysis” (Secretariat: AFNOR, France). The responsible German bodies involved in its preparation were the DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DI

7、N Standards Committee Food and Agricultural Products) and the Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Joint Committee NA 057-05-05 AA Gemeinschaftsausschuss fr die Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standards correspon

8、ding to the International Standards referred to in this document 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 12228:1999-05 as follows: a) the scope of Part 2

9、 of this standard is confined to olive and olive pomace oils. The determination of the contents and composition of sterols in animal and vegetable fats and oils is to be carried out using Part 1 of this standard; b) the precision of the method has been established by an international interlaboratory

10、 test carried out in 2009. The samples used were the following: crude olive pomace oil, refined olive pomace oil, extra virgin olive oil, blend of sunflower oil with extra virgin olive oil and rapeseed oil, and blend of soybean oil with olive oil; c) the standard has been editorially revised. Previo

11、us editions DIN EN ISO 12228: 1999-05 DIN EN ISO 12228-2:2015-02 3 National Annex NA (informative) Bibliography DIN EN ISO 661, Animal and vegetable fats and oils Preparation of test sample DIN EN ISO 5555, Animal and vegetable fats and oils Sampling DIN ISO 3696, Water for analytical laboratory use

12、 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, Accuracy (trueness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatabi

13、lity and reproducibility of a standard measurement method DIN EN ISO 12228-2:2015-02 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 12228-2 October 2014 ICS 67.200.10 Supersedes EN ISO 12228:1999English Version Determination of individual and total sterol

14、s contents - Gas chromatographic method - Part 2: Olive and olive pomace oils (ISO 12228-2:2014) Dtermination de la teneur en strols individuels et totaux -Mthode par chromatographie en phase gazeuse - Partie 2: Huile dolive et huile de grignons dolive (ISO 12228-2:2014)Bestimmung der individuellen

15、und der Gesamtsterine - Gaschromatographisches Verfahren - Teil 2: Oliven- und Oliventresterle (ISO 12228-2:2014) This European Standard was approved by CEN on 6 September 2014. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this E

16、uropean 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 member. This European Standard exists in three official vers

17、ions (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. CEN members are the national standards bodies of Austria,

18、 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, Slovenia, Spain, Sweden, S

19、witzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN nat

20、ional Members. Ref. No. EN ISO 12228-2:2014 EContents PageForeword . 31 Scope . 42 Normative references 43 Terms and definitions . 44 Principle 55 Reagents 56 Apparatus . 67 Sample . 77.1 Sampling . 77.2 Preparation of the test sample 78 Procedure. 78.1 Test portion 78.2 Preparation of unsaponifiabl

21、e matter. 78.3 Separation of the sterol and triterpene dialcohols (erythrodiol, uvaol) fractions by TLC 88.4 Preparation of the trimethylsilyl ethers 98.5 Gas chromatographic analysis . 99 Expression of results . 109.1 Quantitative evaluation 109.2 Determination of the total sterol content . 119.3 C

22、omposition of sterols . 119.4 Composition of triterpene dialcohols . 1110 Precision 1210.1 Interlaboratory test 1210.2 Repeatability limit, r 1210.3 Reproducibility limit, R 1211 Test report 12Annex A (informative) Figures .13Annex B (informative) Interlaborative trial .16Bibliography .19DIN EN ISO

23、12228-2:2015-02 EN ISO 12228-2:2014 (E) 2 Foreword This document (EN ISO 12228-2:2014) 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 of sampling and

24、 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 April 2015, and conflicting national standards shall be withdrawn at the latest by April 2015.

25、 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 12228:1999. According to the CEN-CENELEC Internal Reg

26、ulations, 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, Iceland, Ireland,

27、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 12228-2:2014 has been approved by CEN as EN ISO 12228-2:2014 without any modification. DIN EN

28、 ISO 12228-2:2015-02 EN ISO 12228-2:2014 (E) 3 1 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 s

29、terols in all other animal 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

30、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 vegetable fats and oils Preparation of test sample3 Terms and definitionsFor t

31、he purposes of this document, 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 compos

32、ition is expressed as a 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)

33、, divided by the mass of 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 expresse

34、d as a percentage of all 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 %.DIN EN ISO 12228-2:2015-02 EN ISO 12228-2:2014 (E) 4 4 PrincipleA test portion is saponified by boiling under reflux with

35、an ethanolic 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

36、the 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 sa

37、fety 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

38、 mol/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, appro

39、ximately 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

40、). Take 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.

41、9 Ethanol 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 concentr

42、ation, = 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.DIN EN ISO 12228-2:2015-02 EN ISO 12228-2:2014 (E) 5 5.13 -cholestanol internal standard solution, mass concentration, = 0,2 g/100 ml, in ethyl acetate (5.1

43、0).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 diethyl ether, vol

44、ume 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 disilazane) =

45、 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 ApparatusUsual laborat

46、ory 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 for use with pla

47、tes 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 filtration 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 wi