1、BRITISH STANDARD BS ENISO 15141-2:1998 Foodstuffs Determination of ochratoxin A in cereals and cereal products Part 2: High performance liquid chromatographic method with bicarbonate clean up The European Standard EN ISO 15141-2:1998 has the status of a British Standard ICS 67.060BSENISO15141-2:1998
2、 This British Standard, having been prepared under the directionof the Consumer Products and Services Sector Committee, was published underthe authority of the Standards Committee and comesinto effect on 15 December 1998 BSI 05-1999 ISBN 0 580 30235 0 National foreword This British Standard is the E
3、nglish language version of ENISO15141-2:1998. The UK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis horizontal methods, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/European committee any enqu
4、iries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represented on this panel can be obtained on request to its secretary. Technical Committee AW/4, Ce
5、reals and pulses, has also actively participated in the development of this standard. Cross-references Attention is drawn to the fact that CEN and CENELEC standards normally include an annex which lists normative references to international publications with their corresponding European publications
6、. The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Find” facility of the BSI Standards Electronic Catalogue
7、. 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 legal obligations. Summary of pages This document comprise
8、s a front cover, an inside front cover, pages i and ii, theEN ISO title page, pages 2 to 9 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 on the inside front cover. Amendments issued since
9、publication Amd. No. Date CommentsBSENISO15141-2:1998 BSI 05-1999 i Contents Page National foreword Inside front cover Foreword 2 Text of EN ISO 15141-2 3ii blankEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15141-2 October 1998 ICS 67.060 Descriptors: Food products, cereals, chemical ana
10、lysis, determination of content, ochratoxin, chromatographic analysis, high performance liquid chromatography, extraction, carbonates English version Foodstuffs Determination of ochratoxin A in cereals and cereal products Part2:High performance liquid chromatographic method with bicarbonate clean up
11、 (ISO 15141-2:1998) Produits alimentaires Dosage de lochratoxine A dans les crales et produitsdrivs Partie2:Mthodepar chromatographie liquide haute performance comprenant une tape dextraction par une solution de bicarbonate (ISO15141-2:1998) Lebensmittel Bestimmung von Ochratoxin A in Getreide und G
12、etreideerzeugnissen Teil2:Hochleistungs flssigchromatographisches Verfahren mit Bicarbonatreinigung (ISO 15141-2:1998) This European Standard was approved by CEN on 1 July 1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this E
13、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 Central Secretariat or to any CEN member. This European Standard exists in three official versions (Engl
14、ish, 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 Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, CzechRepub
15、lic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. CEN European Committee for Standardization Comit Europen de Normalisation Europisches Komitee fr Normung Central Secretariat: rue de Stas
16、sart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 15141-2:1998 EENISO15141-2:1998 BSI 05-1999 2 Foreword The text of EN ISO15141-2:1988 has been prepared by Technical Committee CEN/TC275 “Food analys
17、is Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC34 “Agricultural food products”. 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 Ap
18、ril1999, and conflicting national standards shall be withdrawn at the latest by April1999. This European Standard “Foodstuffs Determination of ochratoxin A in cereals and cereal products” consists of two parts: Part 1: High performance liquid chromatographic method with silica gel clean up; Part 2:
19、High performance liquid chromatographic method with bicarbonate clean up. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, CzechRepublic, Denmark, Finland, France, Germa
20、ny, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the UnitedKingdom. Contents Page Foreword 2 1 Scope 3 2 Normative references 3 3 Principle 3 4 Reagents 3 5 Apparatus and equipment 4 6 Procedure 5 7 Calculation 6 8 Precision 6 9 Test repo
21、rt 7 Annex A (informative) Precision data 8 Annex B (informative) Bibliography 9 Table A.1 8 Table A.2 8ENISO15141-2:1998 BSI 05-1999 3 1 Scope This European Standard specifies a method for the determination of ochratoxin A (OTA) at levels greater than34g/kg. The method has been successfully validat
22、ed in interlaboratory studies according to ISO5725:1986 1 on whole barley containing2,94g/kg,3,04g/kg,7,44g/kg and14,44g/kg of ochratoxin A, on whole maize containing8,24g/kg and16,34g/kg of ochratoxin A as well as on wheat bran containing3,84g/kg and4,54g/kg of ochratoxin A. NOTENumerous laboratory
23、 experiences have shown that this method is also applicable to wheat flour. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications ar
24、e listed hereafter. For dated references, subsequent amendments to or revisions of any of these publications apply to this draft European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN ISO 3696, Wat
25、er for analytical laboratory use Specification and test methods (ISO3696:1987). 3 Principle Ochratoxin A is extracted from grains with chloroform-aqueous phosphoric acid and isolated by liquid-liquid partitioning into aqueous bicarbonate solution. The solution is applied to a C 18cartridge, and ochr
26、atoxin A is eluted with ethyl acetate-methanol-acetic acid. Ochratoxin A is separated by reversed phase HPLC and identified and quantified by fluorescence. Chromatography of ochratoxin A methyl ester derivative confirms the identification2 to5. WARNING: Ochratoxin A causes kidney and liver damage an
27、d is a probable carcinogen. Observe appropriate safety precautions6 for handling such compounds and in particular avoid handling in dry form as the electrostatic nature can result in dispersion and inhalation. Glassware can be decontaminated with4% sodium hypochlorite solution. Attention is drawn to
28、 the statement made by the International Agency for Research on Cancer (WHO)7, 8. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade1according to ENISO3696. Solvent shall be of quality for HPLC analysis
29、. 4.1 Chloroform, stabilized with for example2-methyl-2-butene or ethanol 4.2 Phosphoric acid, c(H 3 PO 4 ) . 0,1mol/l 4.3 Diatomaceous earth Soak about900g of acid-washed diatomaceous earth e.g.Celite 545 1)overnight in methanol (4.7). Filter through a double layer of paper in a Buchner funnel (5.6
30、), wash with8l of water and dry for12h at150C. 4.4 Sodium bicarbonate solution, (NaHCO 3 )=30g/l 4.5 Ethyl acetate 4.6 Toluene 4.7 Methanol 4.8 Glacial acetic acid, (CH 3 COOH). 98% 4.9 Acetonitrile 4.10 Dichloromethane 4.11 Elution solution: ethyl acetate (4.5), methanol(4.7) and glacial acetic aci
31、d (4.8) 95+5+0,5 (V + V + V). 4.12 Mobile phase: mix acetonitrile (4.9), water and glacial acetic acid (4.8) 99+99+2 (V + V + V) and degas. 4.13 Solvent mixture: toluene (4.6) and glacial acetic acid (4.8) 99+1 (V + V). 4.14 Boron trifluoride 4.15 Boron trifluoride in methanol solution, (BF 3 )=14g/
32、100ml WARNING: Use a well maintained fume hood. Avoid contact with skin, eyes, and respiratory tract. 4.16 Ochratoxin A, in crystal form or as film in ampoules. 4.17 Ochratoxin A stock solution Dissolve1mg of the ochratoxin A (crystals) (4.16) or the contents of1ampoule (if ochratoxin A has been obt
33、ained as a film) in solvent mixture (4.13) to give a solution containing approximately204g/ml to304g/ml of ochratoxin A. 1) Celite 545 is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endors
34、ement by CEN of these products.ENISO15141-2:1998 4 BSI 05-1999 To determine the exact concentration, record the absorption curve between a wavelength of300nm and370nm in5nm steps in a1cm quartz cell (5.4) with solvent mixture as reference. Identify the wavelength for maximum absorption by recording
35、in1nm steps around the maximum as reference. Calculate the mass concentration of ochratoxin A, ota , in micrograms per millilitre using equation1: where 4.18 Ochratoxin A standard solution Dilute the stock solution (4.17) with solvent mixture(4.13) to obtain a standard solution with a mass concentra
36、tion of ochratoxin A of44g/ml. This solution can be stored in a refrigerator at4C. Stability shall be checked. 4.19 Ochratoxin A calibration solutions Dispense54l, 104l, 254l, 504l and1004l aliquot portions of standard solution(4.18) into separate4ml to5ml vials (5.15) using fixed-volume syringes (5
37、.16). Evaporate just to dryness under nitrogen. Add1,0ml of the mobile phase (4.12) to each vial for final ochratoxin A mass concentrations of0,5ng/254l, 1ng/254l, 2,5ng/254l, 5ng/254l and10ng/254l. 4.20 Sodium hypochlorite solution, (NaOCl)=4g/100ml 5 Apparatus and equipment Usual laboratory equipm
38、ent and, in particular, the following: 5.1 Laboratory mill and a sieve with a1mm aperture size 5.2 High-speed blender. 1250ml jar with cover 5.3 Spectrometer, for measurements of wavelengths between300nm and370nm, having a spectral band width of not more than 2nm 5.4 Quartz cells, with1cm optical pa
39、th length and no significant absorption between wavelengths of300nm and370nm 5.5 Glass fibre filters, 0,3mm thickness, 1,54m pore retention, 9,0cm diameter (or equivalent) 5.6 Buchner funnels, of suitable diameters, e.g.of9cm and25cm 5.7 Fluted filter paper 5.8 Separation funnels, 25ml and100ml 5.9
40、Centrifuge, with100ml tubes or flasks 5.10 Adsorption cartridge, disposable3ml polypropylene tube containing500mg of40m C 18silica 5.11 Vacuum manifold with stopcocks at each port for holding C 18columns. May be replaced by a syringe(5ml to10ml) with a suitable adapter (Luer) 5.12 Test tubes, e.g.10
41、ml with polytetrafluoroethylene (PTFE)-lined screw cap 5.13 Membrane filter, of pore size of approximately0,454m 5.14 HPLC apparatus comprising the following 5.14.1 High performance liquid chromatograph, eluent reservoir, pump with adjustable flow from0,5ml/min to5ml/min, injection valve with e.g.25
42、4l loop, fluorescence detector, compatible recorder or integrator. 5.14.2 HPLC reverse phase analytical column, e.g.from Supelco 2) . NOTEShorter columns can also be used (e.g.a column with a length of120mm to150mm). 5.15 Vials, approximately5ml, with PTFE-lined screw cap, or appropriate sealable co
43、ntainer 5.16 Fixed-volume syringe (1) A max is the absorption determined at the maximum of the absorption curve (here: at333nm) M is the relative molar mass of ochratoxin A (M =403g/mol); 0 is the relative molar absorption coefficient of ochratoxin A in solvent mixture, (here:544m 2 /mol); is the pa
44、th length of the quartz cell in centimetres; length: 250 mm inner diameter: 4,6 mm packing: spherical 5m C 18material or equivalent 2) Supelco is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute a
45、n endorsement by CEN of these productsENISO15141-2:1998 BSI 05-1999 5 6 Procedure 6.1 General The whole analytical procedure should be performed in one working day. If several samples are processed at the same time all samples should be analysed during the following night using an automatic sample i
46、njector. 6.2 Preparation of the test sample Grind the sample and mix it thoroughly until it passes a1mm sieve (5.1) using a laboratory mill(5.1) or mixer and mix thoroughly. 6.3 Extraction of ochratoxin A from the sample Weigh, to the nearest0,1g, a50g test portion prepared as in 6.2 into a blender
47、jar (5.2) and add first250ml of chloroform (4.1) and then25ml of phosphoric acid (4.2). Blend for3min at medium speed. Near the end of blending add10g (45ml) of diatomaceous earth (4.3). Filter the extract through glass fibre paper (5.5) covered with about10g of diatomaceous earth on a9cm Buchner fu
48、nnel (5.6), or through a32cm fluted paper (5.7). Collect at least50ml of filtrate. 6.4 Partition Transfer50ml of the filtrate to a100ml separation funnel (5.8). Add10ml of sodium bicarbonate solution (4.4) and shake gently. Allow the phases to separate. If an emulsion forms, centrifuge for2min at200
49、0min 1 . Collect the upper aqueous phase for cartridge extraction. 6.5 Cartridge preparation Attach the C 18cartridges (5.10) to vacuum manifold ports (5.11) with25ml conical flasks or beakers inside the manifold for collecting conditioning and washing solvents. Wash each cartridge twice with about2ml of methanol (4.7),2ml of water, and2ml of sodium bicarbonate solution (4.4). DO NOT ALLOW THE CARTRIDGE TO RUN DRY. To speed elutions, apply gentle suction. This procedure may also be performed manu
