1、BRITISH STANDARD BS4325-12: 1996 ISO10633-1: 1995 Methods for Analysis of oilseed residues Part12: Determination of glucosinolates content by high-performance liquid chromatography ICS67.200.20BS4325-12:1996 This British Standard, having been prepared under the directionof the Consumer Products and
2、Services Sector Board, was published under the authority of the Standards Boardand comes into effect on 15May1996 BSI08-1999 The following BSI references relate to the work on this standard: Committee reference AW/2 Draft for comment94/501062DC ISBN 0 580 25486 0 Committees responsible for this Brit
3、ish Standard The preparation of this British Standard was entrusted to Technical Committee AW/2, Oilseeds and residues, upon which the following bodies were represented: FOSFA International Grain and Feed Trade Association Leatherhead Food Research Association Ministry of Agriculture, Fisheries and
4、Food National Farmers Union National Institute of Agricultural Botany Seed Crushers and Oil Processors Association Tropical Growers Association United Kingdom Agricultural Supply Trade Association Amendments issued since publication Amd. No. Date CommentsBS4325-12:1996 BSI 08-1999 i Contents Page Co
5、mmittees responsible Inside front cover National foreword ii 1 Scope 1 2 Normative references 1 3 Principle 1 4 Reagents 1 5 Apparatus 1 6 Sampling 2 7 Preparation of test samples 2 8 Procedure 2 9 Expression of results 4 10 Precision 4 11 Test report 5 Annex A (normative) Preparation of reagents 7
6、Annex B (informative) Results of an interlaboratory test 9 Figure 1 Example of a typical chromatogram 6 Table B.1 Determination of glucosinolate content in oilseeds 9 List of references Inside back coverBS4325-12:1996 ii BSI 08-1999 National foreword This Part of BS4325 has been prepared by Technica
7、l Committee AW/2 and is identical with ISO10633-1:1995 Oilseed residues Determination of glucosinolates content Part1: Method using high-performance liquid chromatography, published by the International Organization for Standardization (ISO) and in the preparation of which the United Kingdom played
8、a full part. Additional information. It is recommended that the test sample (clause7) be washed with dichloromethane before continuing with the determination of glucosinolate content (clause8). This is to remove any seed treatment chemicals i.e.pesticides which may be present in the sample and which
9、 may deactivate the enzyme “sulfatase”(4.8). Some pesticides, such as diquat that are used on rapeseed, are not soluble in hexane and will still be in the residue after solvent extraction. Cross-references InternationalStandard Normativereferences Corresponding British Standard ISO771:1977 BS4325 Me
10、thods for analysis of oilseed residues Part1:1978 Determination of moisture and volatile matter content (Technically equivalent) ENISO3696:1995 BSENISO3696:1995 Water for analytical laboratory use. ISO3696:1987 Specification and test methods. (Identical) ISO5502:1992 BS4325 Methods for analysis of o
11、ilseed residues Part9:1992 Preparation of test samples (Identical) ENISO9167-1:1995 ISO9167-1:1992 BSENISO9167 Rapeseed Determination of glucosinolates content Part1:1995 Method using high-performance liquid chromatography (Identical) Informative references ISO5500:1986 BS6606:1987 Methods for sampl
12、ing oilseed residues (Identical) ISO5725:1986 a BS5497 Precision of test methods Part1:1987 Guide for the determination of repeatability and reproducibility for a standard test method by inter-laboratory tests (Identical) a ISO5725:1986, to which informative reference is made in the text, has been s
13、uperseded by ISO5725-1:1994, ISO5725-2:1994, ISO5725-3:1994, ISO5725-4:1994 and ISO5725-6:1994 which are identical with BSISO5725 Accuracy (trueness and precision) of measurement methods and results, BSISO5725-1:1994 General principles and definitions, BSISO5725-2:1994 Basic methods for the determin
14、ation of repeatability and reproducibility of a standard measurement method, BSISO5725-3:1994, Intermediate measures of the precision of a standard measurement method, BSISO5725-4:1994 Basic methods for the determination of the trueness of a standard measurement method, and BSISO5725-6:1994 Use in p
15、ractice of accuracy values.BS4325-12:1996 BSI 08-1999 iii A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from le
16、gal obligations. Summary of pages This document comprises a front cover, an inside front cover, pagesi toiv, pages1to10, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table
17、on the inside front cover.iv blankBS4325-12:1996 BSI 08-1999 1 1 Scope This part of ISO10633 specifies a method for the determination of the content of the different glucosinolates in crucifer oilseeds. NOTE 1This method does not determine glucosinolates which are substituted on the glucose molecule
18、, but these compounds are of little importance in commercial rapeseed. NOTE 2This method allows determination of intact glucosinolates. However, it does not identify and quantify the products formed from the degradation of glucosinolates during preparation of the meal. Therefore, the antinutritional
19、 effects of these degradation products cannot be taken into consideration. 2 Normative references The following standards contain provisions which, through reference in this text, constitute provisions of this part of ISO10633. At the time of publication, the editions indicated were valid. All stand
20、ards are subject to revision, and parties to agreements based on this part of ISO10633 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. ISO771:197
21、7, Oilseed residues Determination of moisture and volatile matter content. ISO3696:1987, Water for analytical laboratory use Specification and test methods. ISO5502:1992, Oilseed residues Preparation of test samples. ISO9167-1:1992, Rapeseed Determination of glucosinolates content Part1: Method usin
22、g high-performance liquid chromatography. 3 Principle Extraction of glucosinolates in a methanol solution, then purification and enzymatic desulfation on ion-exchange resins. Determination using reverse-phase high-performance liquid chromatography (HPLC) with gradient elution and ultraviolet detecti
23、on. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and water complying with the specifications for grade2 of ISO3696. 4.1 Methanol, HPLC grade,70% (V/V) solution. 4.2 Sodium acetate,0,02mol/l solution at pH4,0. 4.3 Sodium acetate,0,2mol/l solution. 4.4 Imida
24、zole formate,6mol/l solution. Dissolve204g of imidazole in113ml of formic acid in a500ml one-mark volumetric flask. Dilute to the mark with water. 4.5 Internal standard Use either sinigrin monohydrate (potassium allylglucosinolate monohydrate, M r=415,49) (seeA.1) or, for rapeseed in which sinigrin
25、is present naturally, glucotropaelin (potassium benzylglucosinolate, M r=447,52) (seeA.2). SeeAnnex A for details of the preparation and purity check of these reagents. 4.6 Mobile phases 4.6.1 Eluant A: water filtered through a0,454m filter and purified by passing through an activated charcoal cartr
26、idge system 1) , or water of equivalent purity. 4.6.2 Eluant B: acetonitrile, HPLC grade,20% (V/V) solution in water that has been purified and passed through a0,454m filter. The concentration may be modified in relation to the column used. 4.7 Ion-exchange resin 4.7.1 DEAE Sepharose CL-6B 2) , sold
27、 as a commercial ready-to-use suspension, or an equivalent product. 4.7.2 DEAE Sephadex A25 2)suspension Mix10g of DEAE SephadexA25 resin (or equivalent) in excess2mol/l acetic acid solution. Leave to settle. Add2mol/l acetic acid until the volume of the supernatant liquid is equal to the volume of
28、the sediment. 4.8 Sulfatase, Helix pomatia typeH1 (EC3.1.6.1) 3) . Purify, test and dilute the sulfatase in accordance with the methods described inA.3.1 toA.3.4. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 High-performance liquid chromatograph, capable of gradient
29、elution and of maintaining a column temperature of30 C, connected to an ultraviolet detector capable of measurements at a wavelength of229nm. 1) The Norganic Millipore system is an example of a suitable product available commercially. This information is given for the convenience of users of this pa
30、rt of ISO10633 and does not constitute an endorsement by ISO of this product. 2) DEAE Sepharose and Sephadex are examples of suitable products available commercially. This information is given for the convenience of users of this part of ISO10633 and does not constitute an endorsement by ISO of thes
31、e products. 3) Sulfatase S-9626 (from Sigma Chemicals) with an activity of16600units/g is an example of a suitable product available commercially. This information is given for the convenience of users of this part of ISO10633 and does not constitute an endorsement by ISO of this product.BS4325-12:1
32、996 2 BSI 08-1999 5.2 Chromatography column for HPLC, type C 18or C 8 , of particle size less than or equal to54m, for example 4) : Lichrosorb RP18 column, 54m (150mm 4,6mm); Spherisorb ODS2 column,u 54m (250mm 4mm;250mm 5mm); Novapak C 18column,u 44m(150mm 4mm); Lichrospher RP8 column,u 54m (125mm
33、4mm); Nucleosil C 18column,u 54m(200mm 4mm). The performance of the column should be checked regularly, preferably using a reference sample of rapeseed desulfoglucosinolate 5) . In particular, the column shall not degrade4-hydroxyglucobrassicin, an important and relatively unstable glucosinolate. Ne
34、w columns shall be subjected to preliminary conditioning in accordance with the manufacturers instructions so that reproducible results can be obtained. 5.3 Double-beam spectrometer, capable of operating in the ultraviolet region of the spectrum, and at a controlled temperature of30 C, equipped with
35、 quartz cells of1cm optical path and a recording system. 5.4 Microgrinder, for example a coffee mill. 5.5 Centrifuge, suitable for use with the tubes(5.6) and capable of obtaining a centrifugal acceleration of5000g. 5.6 Polypropylene tubes, of6ml capacity. 5.7 Water bath, or other heating apparatus,
36、 capable of being maintained at75 C 1 C. 5.8 Glass wool 5.9 Pasteur pipettes,150mm long, and a suitable stand or any other appropriate device. 6 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed during transport or storage
37、. Sampling is not part of the method specified in this part of ISO10633. A recommended sampling method is given in ISO5500 6) . 7 Preparation of test samples Reduce the laboratory sample in accordance with ISO5502 to obtain the required size of test sample. Grind if necessary. Take a sample of this
38、and determine the moisture and volatile matter content in accordance with ISO771. If the result is less than10%(m/m), this value will be used for the calculation(9.1). Continue immediately with the determination of glucosinolate content (clause8) using the test sample without further treatment. If t
39、he moisture and volatile matter content is found to be in excess of10%(m/m), dry the test sample using a current of air at approximately45 C, then redetermine the content. Continue this process until a moisture and volatile matter content of less than10%(m/m) is obtained. This final value is used fo
40、r the calculation. Continue immediately with the determination of glucosinolate content (clause8) using the dried test sample. 8 Procedure NOTE 3If it is required to check whether the repeatability requirement is met, carry out two single determinations in accordance with8.1 to8.4 and8.6 under repea
41、tability conditions. 8.1 Test portion Label two tubes(5.6)A andB and transfer to each a test portion of100mg, weighed to the nearest0,1mg, of the prepared test sample (clause7). 8.2 Extraction of glucosinolates 8.2.1 Place the tubes in the water bath(5.7) set at75 C and leave for1min. Add2ml of boil
42、ing methanol solution(4.1) and then immediately add to tubeA,2004l of5mmol/l internal standard solution(A.1.1); and to tubeB,2004l of20mmol/l internal standard solution(A.1.2). NOTE 4See4.5 for the use of an alternative internal standard solution. 8.2.2 Continue heating at75 C for a further10min, sh
43、aking the tubes at regular intervals. Mix the contents of each tube and then centrifuge at5000g for3min. Transfer the supernatant liquid from each tube to two other tubes(5.6) labelledA andB. 8.2.3 Add, to each of the two tubes A and B containing the solid residue,2ml of boiling methanol(4.1) and re
44、heat in the water bath(5.7) set at75 C for10min, shaking the tubes at regular intervals. 4) The products mentioned are examples of suitable products available commercially. This information is given for the convenience of users of this part of ISO10633 and does not constitute an endorsement by ISO o
45、f these products. 5) Reference samples of oilseed desulfoglucosinolate may be obtained from the Community Reference Bureau (Brussels). 6) ISO5500:1986, Oilseed residues Sampling.BS4325-12:1996 BSI 08-1999 3 Centrifuge for3min and then add the supernatant liquid from the tubesA andB, respectively, to
46、 the tubesA andB, respectively, containing the supernatant liquids retained in8.2.2. 8.2.4 Adjust the volume of the combined extracts in the tubesA andB to approximately5ml with water and mix. These extracts, if stored in the dark in a freezer at18 C, may be kept for2weeks. 8.3 Preparation of ion-ex
47、change columns Cut the required number of Pasteur pipettes(5.9), i.e.two pipettes per sample, so as to leave a volume of1,2ml above the neck and place a glass wool plug(5.8) in the neck of each pipette. Place the pipettes vertically on a stand. Transfer0,5ml of a well-mixed suspension of ion-exchang
48、e resin(4.7.2) to each pipette and allow to settle and drain. Rinse the pipettes with2ml of the imidazole formate(4.4) and then twice with1ml portions of water. 8.4 Purification and desulfation 8.4.1 Carry out the following operations for each combined extract. 8.4.2 Transfer1ml of the extract(8.2.4
49、) to a prepared column(8.3) without disturbing the resin surface and allow to drain. Add two1ml portions of the sodium acetate buffer(4.2), allowing the buffer to drain after each addition. 8.4.3 Add to the column754l of diluted, purified sulfatase solution(4.8). Leave to act overnight at ambient temperature. 8.4.4 Place a tube(5.6) under the column to collect the eluate. Elute the obtained desulfoglucosinolates with two1ml portions of water, allowing the water to drain after each addition. 8.4.5 Mix the eluate well. If not
