1、STD-BSI BS EN IS0 LSL4L-Z-ENGL 1998 Lb24669 0734777 882 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 IS0 15141-2:1998 has the status of a British Standmd ICs 67.06
2、0 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW - BS EN IS0 15 141-2: 1998 BS EN IS0 16141-2:1998 been prepared under the Products and Services Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 December 1998 direction
3、of the Consumer Amd. No. Date O BSI 1998 ISBN O 580 30235 O National foreword Text affected This British Standard is the English language version of EN IS0 1514121998. The LJK participation in its preparation was entrusted to Technical Panel AW/-/3, Food analysis - horizontal methods, which has the
4、responsibility to: - aid enquirers to understand the text; - present to the responsible internationaUEuropean committee any enquiries on the interpretaiion, or proposals for change, and keep the UK interests informed; - monitor related international and European developments and promulgate them in t
5、he UK. A list of organizations represented on this panel can be obtained on request to It3 secretaq Technical Committee AW/4, Cereals 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 normall
6、y include an annex which lists nomiative references to international publications with their corresponding European publications. The British Smdards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section enti
7、tled “International Standards Correspondence Index“, or by using the “Find“ facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of Britkh Standards are responsible for their correct application. Complian
8、ce with a British Standard does not of itself confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN IS0 title page, pages 2 to 12, an inside back cover and a back cover. STD-BSI BS EN IS0 LSL4L-Z-ENGL L778 lbZ4bb9 0734779 655 EU
9、ROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN IS0 15141 -2 October 1998 ICs 67.060 Descriptors: food products, cereals, chemical analysis, detemination of content, ochratoxin, chromatographic analysis, high performance liquid chromatography, extraction, carbonates English version Foodstuffs - D
10、etermination of ochratoxin A in cereais and cereal products - Part 2: High performance liquid chromatographic method with bicarbonate clean up (IS0 151 41 -2:1998) Produits alimentaires - Dosage de Iochratoxine A dans les crales et produits cirivs - Partie 2: Mthode par chromatographie liquide haute
11、 performance comprenant une tape dextraction par une solution de bicamnate (IS0 15141-2:1998) Lebensmittel - Bestimmung von Ochratoxin A in Getreide und Getreideerzeugnissen - Teil 2: Hochieistungs- flssigchromatographisches Verfahren mit Bicarbonatreinigung (IS0 15141-21998) This European Standard
12、was approved by CEN on 1 July 1998. CEN members are bound to comply with the CENKENELEC Intemal Regulations which stipulate the condidons for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nation
13、al standards may be obtained on application to the Central Secretanat 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 CEN member into its own language and notif
14、ied to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland. Ireland, Italy. Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland an
15、d United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMITE EUROPEEN DE NORMALISATION EUROPISCHES KOMITEE FOR NORMUNG Central Secretariat: rue de Sassart, 36 8-1050 Brussels Q 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. E
16、N IS0 15141-21998 E Page 2 EN IC0 15141-2:1998 Contents Foreword . .2 1 Scope . 3 2 Normative references 3 3 Principle 3 4 Reagents .3 5 Apparatus and equipment 5 6 Procedure . 6 7 Calculation 8 8 Precision .9 9 Test repo rt 9 Annex A (informative) Precision data . 1 O Annex B (informative) Bibliogr
17、aphy . .12 Foreword The text of EN IS0 15141-2/1998 has been prepared by Technical Committee CENITC 275 “Food analysis - Horizontal methods“, the secretariat of which is held by DIN, in collaboration with Technical Committee ISOTTC 34 “Agricultural food products“. This European Standard shall be giv
18、en the status of a national standard, either by publication of an identical text or by endorsement, at the latest by April 1999, and conflicting national standards shall be withdrawn at the latest by April 1999. This European Standard ,Foodstuffs - Determination of ochratoxin A in cereals and cereal
19、 products“ consists of two parts: Part 1: High performance liquid chromatographic method with silica gel clean up Part 2: High performance liquid chromatographic method with bicarbonate clean up According to the CENXENELEC Internal Regulations, the national standards organizations of the following c
20、ountries are bound to implement this European Standard: Austria, Belgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Noway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Page 3 EN IS0 15141-2:1998 1 Scope This European
21、Standard specifies a method for the determination of ochratoxin A (OTA) at levels greater than 3 pgikg. The method has been successfully validated in interlaboratory studies according to IS0 5725986 11 on whole barley containing 2,9 pglkg, 3,O pglkg, 7,4 pglkg and 14,4 pglkg of ochratoxin A, on whol
22、e maize containing 8,2 pgikg and 16,3 pgikg of ochratoxin A as well as on wheat bran containing 3,8 pgikg and 4,5 pgikg of ochratoxin A. NOTE: Numerous laboratory experiences have shown that this method is also applicable to wheat flour. 2 Normativa references This European Standard incorporates by
23、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 revisions of any of these publica- tions apply to this draft Europea
24、n Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN IS0 3696 Water for analytical laboratory use - Specification and test methods (IS0 3696:1987) 3 Principle Ochratoxin A is extracted from grains with
25、chloroform-aqueous phosphoric acid and isolated by liquid- liquid partitioning into aqueous bicarbonate solution. The solution is applied to a CIB cartridge, and ochratoxin A is eluted with ethyl acetate-methanol-acetic acid. Ochratoxin A is separated by reversed phase HPLC and identified and quanti
26、fied by fluorescence. Chromatography of ochratoxin A methyl ester derivative confirms the identification 21 to 151. WARNING: Ochratoxin A causes kidney and liver damage and is a probable carcinogen. Ob- serve appropriate safety precautions 161 for handling such compounds and in particular avoid hand
27、ling in dry form as the electrostatic nature can result in dispersion and inhalation. Glass- ware can be decontaminated with 4 % sodium hypochlorite solution. Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO) 71, 81. 4 Reagents During the analysis, unl
28、ess otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1 according to EN IS0 3696. Solvent shall be of quality for HPLC analysis. 4.1 Chloroform, stabilized with for example 2-methyl-2-butene or ethanol 4.2 Phosphoric acid, c(H,PO,) = 0,l mo
29、l/l 4.3 Diatomaceous earth Soak about 900 g of acid-washed diatomaceous earth e.g. Celite 545) overnight in methanol (4.7). Filter through a double layer of paper in a Buchner funnel (5.6), wash with 8 I of water and dry for 12 h at 150 OC. ) Celitem 545 is an example of a suitable product available
30、 commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of these products. STDWBSI BS EN IS0 LSL4L-2-ENGL 1998 = 1624667 0734782 L4T = Page 4 EN IS0 16141-2:1998 4.4 4.5 Ethyl acetate 4.6 Toluene 4.7 Methand 4.8 Glacial ace
31、tic acid, CH,COOH) 98 % 4.9 Acetonitrile 4.1 O Mchloiomethane 4.1 1 Elution solution: ethyl acetate (4.51, methanol (4.7) and glacial acetic acid (4.8) 95 + 5 +0,5 (V+V+v). Sodium bicarbonate solution, p(NaHC0,) = 30 g/l 4.12 Mobile phase: mix acetonitrile (4.91, water and glacial acetic acid (4.8)
32、99 + 99 + 2 (V+ V+ v) and degas. 4.13 Solvent mixture: toluene (4.6) and glacial acetic acid (4.8) 99+ 1 (V+ W. 4.14 Boron trifluoride 4.15 Boron trifluoride in methanol solution, p(BF,) = 14 g/lOO ml WARNING: Use a well maintained fume hood. Avoid contact with skin, eyes, and respiratory tract. 4.1
33、6 Ochratoxin A, in crystal form or as film in ampoules. 4.1 7 Ochratoxin A stock solution Dissolve 1 mg of the ochratoxin A (crystals) (4.16) or the contents of 1 ampoule (if ochratoxin A has been obtained as a film) in solvent mixture (4.13) to give a solution containing approximately 20 pg/ml to 3
34、0pg/ml of ochratoxin A. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps in a 1 cm quartz cell (5.4) with solvent mixture as reference. Identify the wavelength for maximum absorption by recording in 1 nm steps around the maximu
35、m as reference. Calculate the mass concentration of ochratoxin A, pots, in micrograms per millilitre using equation 1 : MX 100 (1) KX6 Polo =Am, where: A, is the absorption determined at the maximum of the absorption curve (here: at 333 nm) M is the relative molar mass of ochratoxin A (M = 403 g/mol
36、); K is the relative molar absorption coefficient of ochratoxin A in solvent mixture, (here: 544 m2/mol); is the path length of the quartz cell in centimetres; 6 STD-BSI BS EN IS0 151q1-2-ENGL 1778 1b24bb7 0734783 086 Page 5 EN IS0 15141-2:1998 4.18 Ochratoxin A standard solution Dilute the stock so
37、lution (4.1 7) with solvent mixture (4.1 3) to obtain a standard solution with a mass concentration of ochratoxin A of 4 pg/ml. This solution can be stored in a refrigerator at 4 OC. Stability shall be checked. 4.1 9 Ochratoxin A calibration solutions Dispense 5 pI, 1 O pl, 25 pl, 50 pI and 1 O0 pi
38、aliquot portions of standard solution (4.18) into separate 4 ml to 5 ml vials (5.15) using fixed-volume syringes (5.16). Evaporate just to dryness under nitrogen. Add 1 ,O ml of the mobile phase (4.12) to each vial for final ochratoxin A mass concentrations of 0,5 ng/25 pl, 1 ng/25 pi, 2,5 ng/25 pl,
39、 5 ng/25 pl and 10 ng/25 pl. 4.20 Sodium hypochlorite solution, p(NaOC1) = 4 g/l O0 mi 5 Apparatus and equipment Usual laboratory equipment and, in particular, the following: 5.1 5.2 5.3 5.4 5.5 5.6 5.7 5.8 5.9 5.10 5.1 1 5.12 5.13 5.14 Laboratory mill and a sieve with a 1 mm aperture size High-spee
40、d blender. 1250 ml jar with cover Spectrometer, for measurements of wavelengths between 300 nm and 370 nm, having a spec- tral band width of not more than i 2 nm Quartz cells, with 1 cm optical path length and no significant absorption between wavelengths of 300 nm and 370 nm Glass fibre filters, 0,
41、3 mm thickness, 1,5 pm pore retention, 9,0 cm diameter (or equivalent) Buchner funnels, of suitable diameters, e. g. of 9 cm and 25 cm Fluted filter paper Separation funnels, 25 ml and 100 mi Centrifuge, with 100 rnl tubes or flasks Adsorption cartridge, disposable 3 ml polypropylene tube containing
42、 500 mg of 40 prn C,* silica Vacuum manifold with stopcocks at each port for holding C, columns. May be replaced by a syringe (5 ml to 10 rnl) with a suitable adapter (Luer) Test tubes, e.g. 1 O ml with polytetrafluoroethylene (PTFE)-lined screw cap Membrane filter, of pore size of approximately 0,4
43、5 pm HPLC apparatus comprising the following 5.14.1 High performance liquid chromatograph, eluent reservoir, pump with adjustable flow from 0,5 ml/rnin to 5 ml/min, injection valve with e.g. 25 pl loop, fluorescence detector, compatible recorder or integrator. Page 6 EN IS0 15141-21998 5.14.2 HPLC r
44、everse phase analytical column, e.g. from Supelco2). length: 250 mm inner diameter: 4,6 mm packing: spherical 5 C,* material or equivalent NOTE: Shorter columns can also be used 1e.g. a column with a length of 120 mm to 150 mm). 5.1 5 Vials, approximately 5 rnl, with PTFE-lined screw cap, or appropr
45、iate sealable container 5.16 Fixed-volume syringe 6 Procedure 6.1 General The whole analytical procedure should be performed in one working day. If several samples are proc- essed at the same time all samples should be analysed during the following night using an automatic sample injector. 6.2 Grind
46、 the sample and mix it thoroughly until it passes a 1 mm sieve (5.1) using a laboratory mill (5.1 1 or mixer and mix thoroughly. Preparation of the test sample 6.3 Extraction of ochratoxin A from the sample Weigh, to the nearest 0,l g, a 50 g test portion prepared as in 6.2 into a blender jar (5.2)
47、and add first 250 ml of chloroform (4.1) and then 25 ml of phosphoric acid (4.2). Blend for 3 min at medium speed. Near the end of blending add 10 g (45 ml) of diatomaceous earth (4.3). Filter the extract through glass fibre paper (5.5) covered with about 10 g of diatomaceous earth on a 9 cm Buchner
48、 funnel (5.61, or through a 32 cm fluted paper (5.7). Collect at least 50 ml of filtrate. 6.4 Partition Transfer 50 mi of the filtrate to a 100 mi separation funnel (5.8). Add 10 ml of sodium bicarbonate solution (4.4) and shake gently. Allow the phases to separate. If an emulsion forms, centrifuge
49、for 2 min at 2000 min-. Collect the upper aqueous phase for cartridge extraction. 6.5 Atta Cartridge preparation h the C, cartridges (5.10) to acuum manifold ports (5.1 1) with 25 ml conical flasks or beakers inside the manifold for collecting conditioning and washing solvents. Wash each cartridge twice with about 2 ml of methanol (4.71, 2 ml of water, and 2 ml 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 manually by applying pressure with a 5 ml to 10 ml s
