DIN EN ISO 12228-1-2014 Determination of individual and total sterols contents - Gas chromatographic method - Part 1 Animal and vegetable fats and oils (ISO 12228-1 2014) German ve.pdf

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1、October 2014Translation by DIN-Sprachendienst.English price group 14No 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

2、 67.200.10!%;wEnglish version EN ISO 12228-1:2014,English translation of DIN EN ISO 12228-1:2014-10Bestimmung der individuellen und der Gesamtsterine Gaschromatographisches Verfahren Teil 1: Tierische und pflanzliche Fette und le (ISO 12228-1:2014);Englische Fassung EN ISO 12228-1:2014,Englische ber

3、setzung von DIN EN ISO 12228-1:2014-10Dtermination de la teneur en strols individuels et totaux Mthode par chromatographie en phase gazeuse Partie 1: Corps gras dorigines animale et vgtale (ISO 12228-1:2014);Version anglaise EN ISO 12228-1:2014,Traduction anglaise de DIN EN ISO 12228-1:2014-10Partia

4、lly supersedesDIN EN ISO 12228:1999-05www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 30 pages09.14 DIN EN ISO 12228-1:2014-102 A comma is used as the decimal marker. National foreword This document (EN ISO 12228-1:2014) has been prepar

5、ed by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France) in collaboration with 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 inv

6、olved in its preparation were the DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products) and the Deutsche Gesellschaft fr Fettwissenschaft (German Fat Research Society), Joint Committee NA 057-05-05 AA Gemein-schaftsausschuss fr die

7、 Analytik von Fetten, len, Fettprodukten, verwandten Stoffen und Rohstoffen. The DIN Standards corresponding 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

8、5725-2 Amendments This standard differs from DIN EN ISO 12228:1999-05 as follows: a) the scope of Part 1 of this standard is confined to animal and vegetable fats and oils. The determination of the contents and composition of sterols in olive and olive pomace oils is to be carried out using Part 2 o

9、f this standard; b) The precision of the method has been established by two international interlaboratory tests organized by Germany in 2011/2012 and 1996/1997. The samples used were the following: refined corn oil, refined sunflower seed oil, refined safflower seed oil, refined rapeseed oil, refine

10、d deodorized bleached palm oil (RDB) and butter fat; c) Annex ZA “Normative references to international publications with their relevant European publications” has been deleted; d) the standard has been editorially revised. Previous editions DIN EN ISO12228: 1999-05 DIN EN ISO 12228-1:2014-10 3 Nati

11、onal 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 Specification and test methods DIN ISO 5725-1, Accuracy (trueness and

12、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 repeatability and reproducibility of a standard measurement method DIN EN ISO 12

13、228-1:2014-10 4 This page is intentionally blank EN ISO 12228-1July 2014 ICS 67.200.10 Supersedes EN ISO 12228:1999English Version Determination of individual and total sterols contents - Gas chromatographic method - Part 1: Animal and vegetable fats and oils (ISO 12228-1:2014) Dtermination de la te

14、neur en strols individuels et totaux -Mthode par chromatographie en phase gazeuse - Partie 1: Corps gras dorigines animale et vgtale(ISO 12228-1:2014) Bestimmung der individuellen und der Gesamtsterine - Gaschromatographisches Verfahren - Teil 1: Tierische und pflanzliche Fette und le (ISO 12228-1:2

15、014) This European Standard was approved by CEN on 14 June 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 r

16、eferences 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 versions (English, French, German). A version in any other language made by translation under the responsibility of a

17、 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, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Maced

18、onia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN A

19、ll rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 12228-1:2014 EEUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMContents Page Foreword .

20、3 1 Scope . 4 2 Normative references. 4 3 Terms and definitions 4 4 Principle 4 5 Reagents . 5 6 Apparatus 6 7 Sample . 6 7.1 Sampling . 6 7.2 Preparation of the test sample 6 8 Procedure 7 8.1 Preparation of the aluminium oxide column . 7 8.2 Test portion . 7 8.3 Extraction of the unsaponifiable ma

21、tter 7 8.4 Thin-layer chromatography . 7 8.5 Isolation of the sterols . 7 8.6 Preparation of sterol trimethylsilyl ethers . 8 8.7 Gas chromatography . 8 9 Expression of results . 8 9.1 Identification of sterols 8 9.2 Composition of sterols 8 9.3 Determination of the total sterol content . 9 10 Preci

22、sion . 10 10.1 Interlaboratory test . 10 10.2 Repeatability limit, r 10 10.3 Reproducibility limit, R 10 11 Test report . 10 Annex A (informative) Figures 11 Annex B (informative) Interlaboratory trial 18 Bibliography 26 2DIN EN ISO 12228-1:2014-10 EN ISO 12228-1:2014 (E) ForewordThis document (EN I

23、SO 12228-1:2014) 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 analysis” the secretariat of which is held by AFNOR. This European St

24、andard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by January 2015, and conflicting national standards shall be withdrawn at the latest by January 2015. Attention is drawn to the possibility that some of the elements o

25、f 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 Regulations, the national standards organizations of the following co

26、untries 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, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

27、Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 12228-1:2014 has been approved by CEN as EN ISO 12228-1:2014 without any modification. 3DIN EN ISO 12228-1:2014-10EN ISO 12228-1:2014 (E)1 ScopeThis part of IS

28、O 12228 specifies a procedure for the gas chromatographic determination of the content and composition of sterols in animal and vegetable fats and oils. However, the determination of the contents and composition of sterols in olive and olive pomace oils is to be carried out using ISO 12228-2.2 Norma

29、tive referencesThe following 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) a

30、pplies.ISO 661, Animal and vegetable fats and oils Preparation of test sampleISO 3696, Water for analytical laboratory use Specification and test methods3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1composition of sterolscomposition of individ

31、ual 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 percentage of all peak areas, normalized to 100 %.3.2total sterol contentmass fraction of the s

32、um 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 of the test portionNote 1 to entry: The content is expressed in milligrams per kilogram.4 Princip

33、leA test portion is saponified by boiling under reflux with an ethanolic potassium hydroxide solution. The unsaponifiable matter is isolated by solid-phase extraction on an aluminium oxide column. The aluminium oxide column is used to retain the fatty acid anions; sterols pass through the column. Th

34、e sterol fraction from the unsaponifiable matter is separated by thin-layer chromatography. The qualitative and quantitative compositions of the sterol fraction are determined by gas chromatography using cholestanol or betulin as the internal standard.4DIN EN ISO 12228-1:2014-10 EN ISO 12228-1:2014

35、(E) 5 ReagentsWARNING Attention is drawn to the regulations which specify the handling of hazardous substances. Technical, organizational, and personal safety measures shall be followed.Use only reagents of recognized analytical grade, unless otherwise stated, and water complying with grade 3 of ISO

36、 36961.5.1 Potassium hydroxide (KOH), ethanolic solution, molar concentration c(KOH) approximately 0,5 mol/l.Dissolve 3 g of potassium hydroxide in 5 ml of water and dilute to 100 ml with ethanol (5.3). The solution should be colourless or straw-coloured.5.2 Internal standard solution, cholestanol (

37、5-cholestan-3-ol) or betulin, volume fraction of 1,0 mg/ml solution in ethanol (see note to 5.10).NOTE In case of hydrogenated oils, which may contain cholestanol, the use of betulin (peak 17 in Table 1) is recommended.5.3 Ethanol, of minimum volume fraction = 95 %.5.4 Aluminium oxide, neutral, part

38、icle size 0,063 mm to 0,200 mm, activity grade I (water content = 0 %).5.5 Diethyl ether, freshly distilled, free from peroxides and residue.WARNING Diethyl ether is highly flammable and can form explosive peroxides. Explosive limits in air are 1,7 % to 48 % (volume fraction). Take special precautio

39、ns when using it. Keep away from heat sources and sunlight.5.6 Silica gel thin-layer chromatography (TLC) plates, commercially available, dimensions 20 cm 20 cm, thickness of layer 0,25 mm.5.7 Developing solvent, hexane/diethyl ether.Volume fraction of each solvent is 50 ml/100 ml.5.8 Standard solut

40、ion for thin-layer chromatography, volume fraction of 1,0 mg/ml cholesterol/cholestanol in acetone or 5,0 mg/ml betulin in acetone.NOTE 1 Cholesterol and cholestanol have the same Rf value (0,35) in TLC while the Rf value for betulin is 0,30 (see Figure A.1).NOTE 2 In case of hydrogenated oils, whic

41、h may contain cholestanol, the use of betulin (peak 17 in Table 1) is recommended.5.9 Spraying reagent, methanol.5.10 Silylating reagent, prepared by adding 50 l of 1-methyl imidazole to 1 ml of N-methyl-N-(trimethylsilyl)-hepta-fluorobutyramide (MSHFBA).NOTE Ready-to-use solutions are commercially

42、available. Other silylation reagents, e.g. bis trimethylsilyl trifluoroacetamide with 1 % trimethylchlorosilane, are also available and can be used when cholestanol is used as internal standard. However for betulin special precautions are taken to ensure that both hydroxyl groups of betulin are sily

43、lated. If not, betulin may show two peaks in the chromatogram.5DIN EN ISO 12228-1:2014-10EN ISO 12228-1:2014 (E)6 ApparatusUsual laboratory apparatus and, in particular, the following.6.1 Round-bottomed flasks, of 25 ml and 50 ml capacity, with ground neck.6.2 Reflux condenser, with ground glass joi

44、nt to fit a flask (6.1).6.3 Glass column, with polytetrafluoroethylene (PTFE) stopper, sintered glass filter and reservoir for 100 ml, length 25 cm, internal diameter 1,5 cm.6.4 Rotary evaporator, attached to a vacuum pump and water bath maintained at 40 C.6.5 Developing tank, made of glass, with a

45、ground glass lid, suitable for use with plates of dimensions 20 cm 20 cm.6.6 Microsyringe, to deliver 100 l.6.7 Oven, maintained at 105 C 3 C.6.8 Desiccator, containing an efficient desiccant, for storing the plates.6.9 Reaction vials, of 0,3 ml to (1,0 to 1,5) ml capacity, with screw caps and PTFE-

46、lined seals, for preparation of sterol derivatives.6.10 Gas chromatograph, for capillary columns, with split injector, flame ionization detector and suitable recorder.6.11 Capillary column, made of fused silica or glass, length 25 m to 60 m, internal diameter 0,2 mm to 0,25 mm, stationary phase SE-5

47、4 (or equivalent non-polar phase with a temperature limit of at least 280 C to 300 C); film thickness about 0,1 m.NOTE A better resolution of the peaks is obtained with a film thickness of 0,1 m.6.12 Microsyringe for gas chromatography, for injecting volumes of 1 l.6.13 Analytical balance, capable o

48、f weighing to the nearest 0,001 g and displaying 0,000 1 g.7 Sample7.1 SamplingSampling is not part of the method specified in this part of ISO 12228. A recommended sampling method is given in ISO 55551.It is important that the laboratory receives a sample which is truly representative and has not b

49、een damaged or changed during transport or storage.7.2 Preparation of the test samplePrepare the test sample in accordance with ISO 661.6DIN EN ISO 12228-1:2014-10 EN ISO 12228-1:2014 (E) 8 Procedure8.1 Preparation of the aluminium oxide columnSuspend 10 g of aluminium oxide (5.4) in 20 ml of ethanol (5.3) and pour the slurry into the glass column (6.3). Allow the aluminium oxide to settle and let the solvent run out of the column