1、BRITISH STANDARDBS EN ISO9167-1:1995BS 4289-9:1993IncorporatingAmendment No. 1Rapeseed Determination ofglucosinolatescontent Part 1: Method using high-performanceliquid chromatographyThe European Standard EN ISO 9167-1:1995 has the status of aBritish StandardI: 995+A1:2013ICS 67.200.20BS EN ISO 9167
2、-1:1995+A1:2013ISBN 978 0 580 79524 4Amendments/corrigenda issued since publicationAmd. No. Date Comments8830 November1995This amendment renumbers BS ISO 9167-1:1992as BS EN ISO 9167-1:1995: Annex ZA added30 September2013Implementation of ISO amendment 1:2013 withCEN endorsement A1:2013: paragraph 1
3、 ofsubclause 9.1 replacedThis British Standard, havingbeen prepared under thedirection of the Agricultureand Food Standards PolicyCommittee, was publishedunder the authority of theStandards Board and comesinto effect on 15 August 1993 The British StandardsInstitution 2013 Published by BSI StandardsL
4、imited 2013 National foreword This British Standard is the UK implementation of EN ISO 9167-1:1995+A1:2013. It is identical to ISO 9167-1:1992, incorporating amendment 1:2013. It supersedes BS EN ISO 9167-1:1995(dual numbered as BS 4289-9:1993), which is withdrawn. Textual errors. When adopting the
5、text of the International Standard thefollowing textual errors were discovered. They have been marked in thetext and have been reported to ISO in a proposal to amend the text of theInternational Standard.In 4.5, paragraph 2, last line, “4.5.2.1” should be read as “4.5.2”.In 4.8.3.2, the concentratio
6、n C, should be read as 1.39 10-4mol/l.In 9.2 and in Figure 1, “desulfoglucobrassicin” (number 15) should be read as“desulfoneoglucobrassicin”.The UK participation in its preparation was entrusted to Technical Committee AW/307, Oilseeds, animal and vegetable fats and oils and theirby-products. A list
7、 of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisions of acontract. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunity fromlegal obl
8、igations.EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 9167-1August 1995ICS 67.200.20Descriptors: Agricultural products, plant products, oilseeds, rapeseeds, chemical analysis, determination of content, glucosinolate, high performance liquid chromatographyEnglish versionRapeseed Determinatio
9、n of glucosinolates content Part 1: Method using high-performance liquid chromatography(ISO 9167-1:1992)Graines de colza Dosage des glusinolates Partie 1: Mthode par chromatographie liquide haute performance(ISO 9167-1:1992)Rapssamen Bestimmung des Glucosinolatgehaltes Teil 1: HPLC-Verfahren(ISO 916
10、7-1:1992)This European Standard was approved by CEN on 1995-05-24. CEN membersare bound to comply with the CEN/CENELEC Internal Regulations whichstipulate the conditions for giving this European Standard the status of anational standard without any alteration.Up-to-date lists and bibliographical ref
11、erences concerning such nationalstandards may be obtained on application to the Central Secretariat or to anyCEN member.This European Standard exists in three official versions (English, French,German). A version in any other language made by translation under theresponsibility of a CEN member into
12、its own language and notified to theCentral Secretariat has the same status as the official versions.CEN members are the national standards bodies of Austria, Belgium,Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy,Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerl
13、andand United Kingdom.CENEuropean Committee for StandardizationComit Europen de NormalisationEuropisches Komitee fr NormungCentral Secretariat: rue de Stassart 36, B-1050 Brussels 1995 All rights of reproduction and communication in any form and by any means reserved in all countries to CEN and its
14、membersRef. No. EN ISO 9167-1:1995 EbersEN ISO 9167-1:1995+A1July 2013EN ISO 9167-1:1995 BSI 03-20002ForewordThe text of the International Standard fromISO/TC 34, “Agricultural food products”, of the International Organization for Standardization (ISO) has been taken over as a European Standard by t
15、he Technical Committee CEN/TC 307, “Oilseeds, vegetables and animal fats and oils and their by-products Methods of sampling and analysis”.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 February 1
16、996, and conflicting national standards shall be withdrawn at the latest by February 1996.According to the CEN/CENELEC Internal Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Lu
17、xembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.ContentsPageForeword 21 Scope 32 Normative references 33 Principle 34 Reagents 35 Apparatus 56 Sampling 67 Preparation of the test sample 68 Procedure 69 Expression of results 710 Precision 811 Test report 8Annex
18、A (informative) Bibliography 10Annex ZA (normative) Normative references to international publications with their relevant European publications 10Figure 1 Example of a typicalchromatogram 9Table 1 Statistical results ofinter-laboratory test 8EN ISO 9167-1:1995 BSI 03-20002ForewordThe text of the In
19、ternational Standard fromISO/TC 34, “Agricultural food products”, of the International Organization for Standardization (ISO) has been taken over as a European Standard by the Technical Committee CEN/TC 307, “Oilseeds, vegetables and animal fats and oils and their by-products Methods of sampling and
20、 analysis”.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 February 1996, and conflicting national standards shall be withdrawn at the latest by February 1996.According to the CEN/CENELEC Internal
21、 Regulations, the following countries are bound to implement this European Standard: Austria, Belgium, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.ContentsPageForeword 21 Scope 32 Normati
22、ve references 33 Principle 34 Reagents 35 Apparatus 56 Sampling 67 Preparation of the test sample 68 Procedure 69 Expression of results 710 Precision 811 Test report 8Annex A (informative) Bibliography 10Annex ZA (normative) Normative references to international publications with their relevant Euro
23、pean publications 10Figure 1 Example of a typicalchromatogram 9Table 1 Statistical results ofinter-laboratory test 8BS EN ISO 9167-1:1995+A1:2013EN ISO 9167-1:1995+A1:2013 The British Standards Institution 2013Foreword to amendment A1ISO (the International Organization for Standardization) is a worl
24、dwide 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 represented on
25、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 develop this do
26、cument 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 ISO/IEC Direct
27、ives, Part 2, 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 the development o
28、f the document will be in the Introduction and/or on the ISO list of patent declarations received, 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.The committee responsible for this document is ISO/TC 3
29、4, Food products, Subcommittee SC 2, Oleaginous seeds and fruits and oilseed meals.EN ISO 9167-1:1995 BSI 03-2000 31 ScopeThis part of ISO 9167 specifies a method for the determination of the content of the different glucosinolates in rapeseeds (colza) usinghigh-performance liquid chromatography.NOT
30、E 1 This method does not determine glucosinolates which are substituted on the glucose molecule, but these compounds are of little importance in commercial rapeseed.NOTE 2 A rapid method for the determination of glucosinolates content using X-ray fluorescence spectrometry is the subject of ISO 9167-
31、2.2 Normative referencesThe following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO 9167. At the time of publication, the editions indicated were valid. All standards are subject to revision, and parties to agreements based on this part
32、 of ISO 9167 are encouraged to investigate the possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain registers of currently valid International Standards.ISO 664:1990, Oilseeds Reduction of laboratory sample to test sample. ISO 665:1977, O
33、ilseeds Determination of moisture and volatile matter content. ISO 3696:1987, Water for analytical laboratory use Specification and test methods. 3 PrincipleExtraction of glucosinolates by methanol, then purification and enzymatic desulfatation onion-exchange resins. Determination usingreversed-phas
34、e high-performance liquid chromatography (HPLC) with elution gradient and ultraviolet detection.4 ReagentsUse only reagents of recognized analytical grade, unless otherwise specified, and water complying with grade 2 of ISO 3696.4.1 Methanol, HPLC grade, 70 % (V/V) solution.4.2 Sodium acetate, 0,02
35、mol/l at pH 4,0.4.3 Sodium acetate, 0,2 mol/l solution.4.4 Imidazole formate, 6 mol/l solution.Dissolve 204 g of imidazole in 113 ml of formic acid in a 500 ml one-mark volumetric flask. Make up to the mark with water.4.5 Internal standard, use either sinigrin monohydrate (potassium allylglucosinola
36、te monohydrate, Mr= 415,49) (see 4.5.1) or, for rapeseed (cultivated or self-propagated) in which sinigrin is present naturally, glucotropaeolin (benzylglucosinolate, potassium salt, Mr= 447,52) (see 4.5.2).For rapeseed with a low glucosinolate content ( 20 4m/g), reduce the internal standard concen
37、tration (1 mmol/l to3mmol/l) in 4.5.1 and 4.5.2.1.4.5.1 Sinigrin monohydrate4.5.1.1 Sinigrin monohydrate, 5 mol/l solution.Dissolve 207,7 mg of potassium allylglucosinolate monohydrate in water in a 100 ml one-mark volumetric flask. Make up to the mark with water.The solution thus prepared may be st
38、ored in a refrigerator at approximately4C for up to a week or in a freezer at18 C for a longer period.4.5.1.2 Sinigrin monohydrate, 20 mmol/l solution.Dissolve 831,0 mg of potassium allylglucosinolate monohydrate in water in a 100 ml one-mark volumetric flask. Make up to the mark with water.The solu
39、tion thus prepared may be stored in a refrigerator at approximately4C for up to a week or in a freezer at18 C for a longer period.4.5.1.3 Purity checkUse one or more of the following three tests: HPLC analysis using the method specified in this part of ISO 9167; analysis of the intact sinigrin by HP
40、LC(ion-pair technique); analysis of the desulfated and silylated sinigrin by gas chromatography.For each test, the chromatogram shall show only one major peak representing at least 98 % of the total peak area.Confirm the purity by determining the quantity of glucose released after hydrolysis with my
41、rosinase (thioglucoside glucohydrolase, EC 3.2.3.1). Measure the glucose by enzymatic means. The use of a commercially available test kit facilitates the determination. Take into account any free glucose present; this is determined in the same way but without addition of myrosinase. The molar concen
42、tration of glucose measured should be at least 98 % of the molar concentration of the sinigrin solution tested.BS EN ISO 9167-1:1995+A1:2013EN ISO 9167-1:1995+A1:20133 The British Standards Institution 2013EN ISO 9167-1:19954 BSI 03-20004.5.2 GlucotropaeolinNOTE 3 Glucotropaeolin is sometimes diffic
43、ult to separate from other natural minor glucosinolates.4.5.2.1 Glucotropaeolin, 5 mmol/l solution.Dissolve 233,8 mg of glucotropaeolin in water in a 100 ml volumetric flask. Make up to the mark with water.4.5.2.2 Glucotropaeolin, 20 mmol/l solution.Dissolve 895,0 mg of glucotropaeolin in water in a
44、 100 ml volumetric flask. Make up to the mark with water.4.5.2.3 Purity checkCheck the purity in accordance with the procedure described in 4.5.1.3.4.5.2.4 Response factorVerify that the response factors of glucotropaeolin, in comparison with sinigrin, correspond to those indicated in 9.2.4.6 Mobile
45、 phases4.6.1 Eluant A: water, purified by passing it through an activated charcoal cartridge (e.g. Norganic Millipore1)system) or water of equivalent purity.4.6.2 Eluant B: acetonitrile, HPLC grade, 20 % (V/V) solution in purified water. The concentration may be modified in relation to the column us
46、ed.4.7 Ion-exchange resin, use either 4.7.1 or 4.7.2.4.7.1 DEAE Sepharose CL-6B2)suspension, available commercially ready for use, or an equivalent product.4.7.2 DEAE Sephadex A252)suspension, prepared as follows.Mix 10 g of DEAE Sephadex A25 resin (or an equivalent resin) in excess2mol/l acetic aci
47、d solution. Leave to settle. Add2mol/l acetic acid until the volume of the liquid is equal to twice the volume of the sediment.4.8 Sulfatase, Helix pomatia type H1 (EC 3.1.6.1), having an activity of greater than 0,5 units of activity per millilitre of purified sulfatase solution.Purify, test and di
48、lute the sulfatase in accordance with the method described in 4.8.1 to 4.8.4.4.8.1 Preparation of ion-exchange columnsCut five Pasteur pipettes (5.9)7cm above the neck and place a glass wool plug (5.8) in the neck. Place the pipettes vertically on a stand and add to each a sufficient quantity of ion
49、-exchange resin (4.7) such that, once the water has drained off, a volume of 500 4l of resin is obtained.Pour1ml of the imidazole formate solution (4.4) into each pipette and rinse twice with1ml portions of water.4.8.2 PurificationWeigh, to the nearest 0,1 mg, 25 mg of Helix pomatia type H1 (4.8), dissolve it in 2,5 ml of water and transfer 500 4l of this solution to each of the columns prepared in 4.8.1. Wash each column with 1,5 ml of water and discard the effluent. Then add 1,5 ml of the sodium acetate solution (4.3) and collect the
