1、BRITISH STANDARD BS EN ISO 15141-1:1998 Foodstuffs Determination of ochratoxin A in cereals and cereal products Part 1: High performance liquid chromatographic method with silica gel clean up The European Standard EN ISO 15141-1:1998 has the status of a BritishStandard ICS 67.060BSENISO15141-1:1998
2、This British Standard, having been prepared under the directionof the Consumer Products and Services SectorCommittee, was publishedunderthe authorityofthe Standards Committee and comes into effect on 15December1998 BSI 05-1999 ISBN 0 580 30234 2 National foreword This British Standard is the English
3、 language version of EN ISO15141-1:1998. 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 enquiries
4、 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, Cereals
5、 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. The
6、 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. A B
7、ritish 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 comprises a f
8、ront cover, an inside front cover, pages i and ii, theEN ISO title page, pages 2 to 10 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 publ
9、ication Amd. No. Date CommentsBSENISO15141-1:1998 BSI 05-1999 i Contents Page National foreword Inside front cover Foreword 2 Text of EN ISO 15141-1 3ii blankEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15141-1 October 1998 ICS 67.060 Descriptors: Food products, cereals, chemical analysi
10、s, determination of content, ochratoxin, chromatographic analysis, high performance liquid chromatography, extraction, silicon dioxid English version Foodstuffs Determination of ochratoxin A in cereals andcereal products Part1:High performance liquid chromatographic method with silica gel clean up (
11、ISO 15141-1:1998) Produits alimentaires Dosage de lochratoxine A dans les crales et produitsdrivs Partie1:Mthodeparchromatographie liquidehaute performance comprenant une tape dextraction par chromatographie sur gelde silice (ISO 15141-1:1998) Lebensmittel Bestimmung von Ochratoxin A in Getreide und
12、 Getreideerzeugnissen Teil1:Hochleistungs- flssigchromatographisches Verfahren mit Kieselgelreinigung (ISO 15141-1:1998) 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
13、 European 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 (En
14、glish, 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, CzechRep
15、ublic, 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 St
16、assart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwidefor CEN national Members. Ref. No. EN ISO 15141-1:1998 EENISO15141-1:1998 BSI 05-1999 2 Foreword The text of EN ISO15141-1:1998 has been prepared by Technical Committee CEN/TC275 “Food analy
17、sis 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 A
18、pril 1999, and conflicting national standards shall be withdrawn at the latest by April 1999. This European Standard “Foodstuffs Determination of ochratoxin A in cereal 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, Ger
20、many, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and theUnitedKingdom. 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 7 8 Precision 7 9 Test rep
21、ort 8 Annex A (informative) Precision data 9 Annex B (informative) Bibliography 10 Table A.1 9ENISO15141-1:1998 BSI 05-1999 3 1 Scope This European Standard specifies a method for the determination of ochratoxin A at levels greater than0,44g/kg. The method has been successfully validated in2interlab
22、oratory studies according to ISO5725:1996 1 on wheat whole meal containing0,44g/kg and1,24g/kg of ochratoxin A. NOTENumerous laboratory experiences have shown that this method is also applicable to cereals, dried fruits, oilseeds, pulses, wine, beer, fruit juices and raw coffee, see 2, 3, 4. 2 Norma
23、tive references This draft 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 are listed hereafter. For dated references, subsequent amendments to or revisio
24、ns 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 3696:1995, Water for analytical laboratory use Specification and test methods (ISO3696
25、:1987). 3 Principle Ochratoxin A (OTA) is extracted with toluene after acidification with hydrochloric acid and after the ionic strength has been increased by adding magnesium chloride. The extract is purified using a mini silica gel column and ochratoxin A is determined by high performance liquid c
26、hromatography (HPLC) on a reversed phase column and identified and modified by fluorescence. The result is verified, if required, by derivatization with boron trifluoride in methanolic solution 5, 6. WARNING: Ochratoxin A causes kidney and liver damage and is a probable carcinogen. Observe appropria
27、te safety precautions 7 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 the statement made by the International Age
28、ncy for Research on Cancer(WHO) 8, 9. 4 Reagents During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade1 according to ENISO3696. Solvent shall be of quality for HPLC analysis. 4.1 Sodium sulfate, anhydrous 4.2 Glacial
29、 acetic acid (CH 3 COOH) . 98% 4.3 Solution of hydrochloric acid c(HCl) = 2 mol/l 4.4 Magnesium chloride solution c(MgCl 2 )=0,4mol/l 4.5 Acetonitrile 4.6 Toluene 4.7 n-Hexane 4.8 Dichloromethane 4.9 Acetone 4.10 Methanol 4.11 Solvent mixture I: toluene (4.6) and glacial acetic acid (4.2) 99 + 1 par
30、ts per volume (V + V) 4.12 Solvent mixture II: acetone (4.9) and toluene(4.6) 5 + 95 (V + V) 4.13 Solvent mixture III: toluene (4.6) and glacial acetic acid (4.2) 90 + 10 (V + V) 4.14 Mobile phase Mix99volume parts of acetonitrile (4.5) with99volume parts of water and2volume parts of glacial acetic
31、acid (4.2) and degas this solution before use. 4.15 Boron trifluoride 4.16 Boron trifluoride in methanol solution, (BF 3 )=14 g/100 mlENISO15141-1:1998 4 BSI 05-1999 WARNING: Use a well maintained fume hood. Avoid contact with skin, eyes, and respiratory tract. 4.17 Ochratoxin A, in crystal form or
32、as a film in ampoules 4.18 Ochratoxin A stock solution Dissolve1mg of the ochratoxin A (crystals) (4.17) or the contents of1ampoule (if ochratoxin A has been obtained as a film) in solvent mixture I (4.11) to give a solution containing approximately204g/ml to 304g/ml of ochratoxin A. To determine th
33、e exact concentration, record the absorption curve between a wavelength of300nm and370nm in5nm steps in a1cm quartz cell (5.5) with solvent mixture I (4.11) as reference. Identify the wavelength for maximum absorption by recording in1nm steps around the maximum as reference. Calculate the mass conce
34、ntration of ochratoxin A, OTA , in micrograms per millilitre of solution using equation 1: where 4.19 Ochratoxin A standard solution(OTA)=1 4g/ml Evaporate under a nitrogen flow1ml of the stock solution (4.18) or the aliquot portion which is equivalent to an absolute amount of1004g of Ochratoxin A t
35、o dryness and dilute to100ml with the mobile phase(4.14). This solution can be stored in a refrigerator at4C. Stability shall be checked. 4.20 Ochratoxin A calibration solutions Pipette suitable volumes of ochratoxin A standard solution (4.19), e.g.1ml, 2,5ml,4ml and5ml into e.g.a100ml volumetric fl
36、ask (5.12) and dilute to the mark with the mobile phase (4.14). The amount of ochratoxin A in the calibration solutions should cover the range of0,2ng to1,0ng per20-l-injection volume. 4.21 Sodium hypochlorite solution, (NaOCl) =4g/100 ml 5 Apparatus and equipment Usual laboratory equipment and, in
37、particular, the following: 5.1 Laboratory mill, suitable to grind to1mm 5.2 Rotary evaporator, with a water bath capable of being controlled between20C and50C 5.3 Mechanical shaker 5.4 Spectrometer, suitable for measurement at wavelengths of300nm up to370nm, having a spectral band width of not more
38、than 2nm 5.5 Quartz cells, with1cm optical path length and no significant absorption between wavelengths of300nm and370nm 5.6 Centrifuge tubes, e.g.of capacity250ml, plastic made of high density polyethylene (HDPE), with screw cap 5.7 Cooling centrifuge, preferably a refrigerated centrifuge, capable
39、 of producing a gravitational force of at least3500g at the base of the centrifuge tubes (5.6) (1) A max is the absorption determined at the maximum of the absorption curve (here:at333nm); M is the relative molecular mass of ochratoxin A (M = 403 g/mol); is the molar absorption coefficient of ochrat
40、oxin A, in solvent mixture I (here:544 m 2 /mol); is the path length of the cell in centimetres.ENISO15141-1:1998 BSI 05-1999 5 5.8 Solid phase extraction columns, e.g.SEP-PAK 1)disposable silica gel After the pack has been opened, condition at105C for2h and store over activated silica gel with mois
41、ture indicator. Before use, wash with10ml of toluene (4.6). Check the recovery with each new batch. In the case of use of SEP-PAK columns, the cartridges have the following specification: in a3ml polypropylene tube. 5.9 Solvent containers, such as syringes, e.g.of50ml capacity with central opening a
42、nd stop-cock 5.10 Pear-shaped flasks,50ml, with ground glassjoint 5.11 Separating funnel, 50 ml 5.12 Volumetric flask, 100 ml 5.13 Membrane filter for aqueous solutions, made of polytetrafluoroethylene (PTFE), with a diameter of4mm and a pore size of0,45 4m 5.14 Sieve, with an aperture size of not m
43、ore than1mm 5.15 Vials with crimped caps or screw cap vials 5.16 Microsyringe, of capacity5004l 5.17 HPLC apparatus, comprising the following 5.17.1 High performance liquid chromatograph, eluent reservoir, a pump, an injection system, a fluorescence detector with variable wavelength setting and a da
44、ta processing, e.g.an integrator with plotter. 5.17.2 Analytical reversed phase HPLC separating column, C 18 , e.g.Lichrospher100RP18 2)which ensures a baseline resolved resolution of the ochratoxin A peak from all other peaks. NOTEShorter columns can also be used (e.g.a column with a length of120mm
45、 to150mm) 5.17.3 Precolumn, C 18 , 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 injector. 6.2 Preparation of the tes
46、t samples Grind the laboratory sample using a laboratory mill(5.1) until it passes through the sieve (5.14) and mix it thoroughly. NOTEGrinding is not necessary for wheat flour with a maximum size of2504m. 1) SEP PAK is an example of a suitable product available commercially. This information is giv
47、en for the convenience of users of this Standard and does not constitute an endorsement by CEN of these products. mean mass of the packing material: 690 mg pore size: 12,5 nm particle size: 55 4m to 105 4m 2) Lichrospher 100 RP 18 is an example of a suitable product available commercially. This info
48、rmation is given for the convenience of users of this Standard and does not constitute an endorsement by CEN of these products. length: 250 mm internal diameter: 4 mm spherical particles of size: 5 4m length: 40 mm internal diameter: 4 mm spherical particles of size: 5 4mENISO15141-1:1998 6 BSI 05-1
49、999 6.3 Extraction of ochratoxin A from the sample Place20g (m 0 ), weighed to the nearest0,1g, of the sample prepared as in 6.2 in a centrifuge tube (5.6). For ochratoxin A contents of more than5,0 4g/kg repeat the analysis using a test portion of10g, otherwise the risk of reduced recovery has to be taken into account. Successively add30ml of hydrochloric acid solution (4.3),50ml of magnesium chloride solution (4.4), stir with a glass rod, and add100ml of toluene(4.6) (V 1 )
