DIN EN ISO 16050-2011 Foodstuffs - Determination of aflatoxin B and the total content of aflatoxins B B G and G in cereals nuts and derived products - High performance liquid chrom.pdf

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1、September 2011 Translation by DIN-Sprachendienst.English price group 11No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).

2、ICS 67.060; 67.080.10!$tk(“1817205www.din.deDDIN EN ISO 16050SupersedesDIN EN 12955:1999-09www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises pages0 .11Foodstuffs Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G

3、1and G2in cereals, nuts and derived products High performance liquid chromatographic method (ISO 16050:2003) English translation of DIN EN ISO 16050:2011-09 Lebensmittel Bestimmung von Aflatoxin B1und der Summe von Aflatoxin B1, B2, G1und G2in Getreiden, Nssen und verwandten Produkten Hochleistungsf

4、lssigchromatographisches Verfahren (ISO 16050:2003) Englische bersetzung von DIN EN ISO 16050:2011-09 Produits alimentaires Dosage de laflatoxine B1et dtermination de la teneur totale en aflatoxines B1, B2, G1et G2dans les crales, les fruits coque et les produits drivs Mthode par chromatographie liq

5、uide haute performance (ISO 16050:2003) Traduction anglaise de DIN EN ISO 16050:2011-09 179 DIN EN ISO 16050:2011-09 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products”, (Secretariat: AFNOR/ ABNT, France/Brazil), S

6、ubcommittee SC 4 “Cereals and pulses” (Secretariat: SAC, China) in collaboration with CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agr

7、icultural Products Standards Committee), Working Committee NA 057-01-03 AA Biotoxine. Originally, EN 12955:1999, Foodstuffs Determination of aflatoxin B1, and the sum of aflatoxins B1, B2, G1and G2in cereals, shell-fruits and derived products High performance liquid chromatographic method with post

8、column derivatization and immunoaffinity column clean up was the basis for ISO 16050:2003. The DIN Standard corresponding to the Inernational Standard referred to in this document is as follows: ISO 3696 DIN ISO 3696 Amendments This standard differs from DIN EN 12955:1999-09 as follows: a) the metho

9、d is identical to that specified in EN 12955 and has been adopted by ISO as ISO 16050. The text of the standard has been editorially revised. Previous editions DIN EN 12955: 1999-09 National Annex NA (informative) Bibliography DIN ISO 3696, Water for analytical laboratory use Specification and test

10、methods 2 EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 16050 July 2011 ICS 67.060; 67.080.10 Supersedes EN 12955:1999English Version Foodstuffs - Determination of aflatoxin B1, and the total content of aflatoxins B1, B2, G1and G2in cereals, nuts and derived products - High-performance li

11、quid chromatographic method (ISO 16050:2003) Produits alimentaires - Dosage de laflatoxine B1 et dtermination de la teneur totale en aflatoxines B1, B2, G1 et G2 dans les crales, les fruits coque et les produits drivs - Mthode par chromatographie liquide haute performance (ISO 16050:2003) Lebensmitt

12、el - Bestimmung von Aflatoxin B1 und der Summe von Aflatoxin B1 , B2 , G1 und G2 in Getreiden, Nssen und verwandten Produkten - Hochleistungsflssigchromatographisches Verfahren This European Standard was approved by CEN on 17 June 2011. CEN members are bound to comply with the CEN/CENELEC Internal R

13、egulations which stipulate the conditions for giving this 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 CEN-CENELEC Management Centre or to any CEN m

14、ember. 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 notified to the CEN-CENELEC Management Centre has the same status as the official versions

15、. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Sloven

16、ia, Spain, Sweden, Switzerland and United Kingdom. Management Centre: Avenue Marnix 17, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 16050:2011: E(ISO 16050:2003) EUROPEAN COMMITTEE FOR STANDARDIZATION C

17、OMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Contents Page Foreword. 3 1 Scope 4 2 Normative references . 4 3 Principle . 4 4 Reagents 4 5 Apparatus 7 6 Procedure 8 6.1 General. 8 6.2 Extraction. 8 6.3 Clean-up. 9 6.4 HPLC operating conditions 9 6.5 Identification 9 6.6 Calibration g

18、raph 9 6.7 Determination 10 7 Calculation of results 10 8 Precision 11 8.1 Interlaboratory test . 11 8.2 Repeatability 11 8.3 Reproducibility 12 9 Test report . 12 Annex A (informative) Results of interlaboratory test 13 Bibliography . 15 2DIN EN ISO 16050:2011-09 EN ISO 16050:2011 (E) Foreword The

19、text of ISO 16050:2003 has been prepared by Technical Committee ISO/TC 34 “Food products” of the International Organization for Standardization (ISO) and has been taken over as EN ISO 16050:2011 by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held b

20、y DIN. 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 January 2012, and conflicting national standards shall be withdrawn at the latest by January 2012. Attention is drawn to the possibility that

21、 some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes EN 12955:1999. Originally, EN 12955:1999 “Foodstuffs - Determination of aflatoxin B1, and the sum of af

22、latoxins B1, B2, G1and G2in cereals, shell-fruits and derived products - High performance liquid chromatographic method with post column derivatization and immunoaffinity column clean up“ was the basis for ISO 16050. In order to avoid having two equal standards on CEN- and ISO-level on the same topi

23、c, it was decided to take over ISO 16050 as EN ISO 16050 and to withdraw EN 12955 as soon as EN ISO 16050 is published. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium,

24、Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement no

25、tice The text of ISO 16050:2003 has been approved by CEN as a EN ISO 16050:2011 without any modification. 3DIN EN ISO 16050:2011-09 EN ISO 16050:2011 (E) WARNING The use of this standard involves hazardous materials and operations. This standard does not purport to address all the safety problems as

26、sociated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practice and to determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard specifies a reverse-phase high-performance liquid chromatogr

27、aphic method, with immunoaffinity column clean-up and post-column derivatization, for the determination of aflatoxins in cereals, nuts and derived products. The limit of quantification for aflatoxin B1, and for the sum of aflatoxins B1, B2, G1and G2, is 8 g/kg. The method has been validated for maiz

28、e containing 24,5 g/kg, for peanut butter containing 8,4 g/kg, and for raw peanuts containing 16 g/kg of total aflatoxins. It has also been shown that this method can be used for oilseed products, dried fruits and derived products. 2 Normative references The following referenced documents are indisp

29、ensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 3696:1987, Water for analytical laboratory use Specification and test methods 3 Principle

30、The test sample is extracted with a mixture of methanol and water. The sample extract is filtered, diluted with water, and applied to an affinity column containing antibodies specific for aflatoxins B1, B2, G1and G2. The aflatoxins are isolated, purified and concentrated on the column then removed f

31、rom the antibodies with methanol. The aflatoxins are quantified by reverse-phase high-performance liquid chromatography (HPLC) with fluorescence detection and post-column derivatization. 4 Reagents Use only reagents recognized analytical grade, unless otherwise stated. 4.1 Water, according to grade

32、1 of ISO 3696:1987. 4.2 Sodium chloride. 4DIN EN ISO 16050:2011-09 EN ISO 16050:2011 (E) 4.3 Iodine, crystalline, or as an alternative, pyridinium hydrobromide perbromide (PBPB)1). 4.4 Aflatoxin, in crystal form or as a film ampoule. WARNING Aflatoxins are carcinogenic to human subjects. Attention i

33、s drawn to the statement made by the International Agencies for Research on Cancer (WHO) (see 1, 2). Adequately protect from daylight the laboratory where the analyses are carried out. This may be achieved effectively by using ultraviolet (UV) absorbing foil on the windows in combination with subdue

34、d light (no direct sunlight), or curtains or blinds in combination with artificial light (fluorescent tubes are acceptable). 4.5 Acetonitrile, HPCL grade. 4.6 Methanol, analytical grade. 4.7 Methanol, HPLC grade. 4.8 Toluene, analytical grade. WARNING Toluene is highly flammable and harmful. Standar

35、d preparation involving this solvent shall be performed in a fume cupboard. Operations outside the fume cupboard, such as measurement of standards by UV spectrometry, shall be performed with the standards in closed containers. 4.9 Toluene/acetonitrile mixture Mix 98 parts per volume of toluene (4.8)

36、 with 2 parts per volume of acetonitrile (4.5) (see Warning in 4.8). 4.10 Extraction solvent Mix 7 parts per volume of methanol (4.6) with 3 parts per volume of water (4.1). Other extraction solvent mixtures which are compatible with the mobile phase may also be used if proved to be more effective o

37、r recommended by the manufacturer of the immunoaffinity (IA) column. 4.11 Mobile phase Mix 3 parts per volume of water (4.1) with 1 part per volume of acetonitrile (4.5) and 1 part per volume of methanol (4.7). Degas the solution before use. 4.12 Post-column derivatization reagent Dissolve 100 mg of

38、 iodine (4.3) in 2 ml of methanol (4.6). Add 200 ml of water (4.1), stir for 1 h, then filter through a 0,45 m membrane filter (5.8). Prepare the solution the week of use and store the solution in the dark or in a brown glass bottle. Before use, stir the solution for 10 min. As an alternative, disso

39、lve 50 mg of PBPB (4.3) in 1 000 ml of water. This solution may be used for up to 4 days if stored in a dark place at room temperature. 4.13 Aflatoxin B1, B2, G1and G2stock solutions WARNING Protect solutions containing aflatoxin from light as far as possible (keep in the dark, use aluminium foil or

40、 amber-coloured glassware). 1) CAS: 39416-48-3 (CAS = Chemical Abstract Service). 5DIN EN ISO 16050:2011-09 EN ISO 16050:2011 (E) Dissolve aflatoxin B1, B2, G1and G2separately in the toluene/acetonitrile mixture (4.9) to give separate solutions containing 10 g/ml.To determine the exact concentration

41、 of aflatoxin in each stock solution, record the absorption curve at a wavelength between 330 nm and 370 nm in 1 cm quartz glass cells (5.7) using a spectrometer (5.6) with a toluene/acetonitrile mixture (4.9) as reference. Calculate the aflatoxin concentration of each aflatoxin, g85i, in micrograms

42、 per millilitre, using Equation (1): max1000iiiAMdg85g72g117g117g32g117(1) whereAmaxis the absorbance determined at the maximum of the absorption curve; Miis the molecular mass of each aflatoxin, in grams; g72iis the molar absorption coefficient of each aflatoxin in toluene/acetonitrile; NOTE This v

43、alue is determined in a solution that contains c = 1 mol/l of aflatoxin and in a cell with the optical pathlength d = 1 cm. The molar absorption coefficient (g72) is usually given without a unit of measurement, but from the equation A = g72 g117 c g117 d, the following unit can be derived for it: lg

44、185molg161g185cmg161. d is the optical pathlength of the cell, in centimetres. Miand g72iare given in Table 1.Table 1 Molecular mass and molar absorption coefficient of aflatoxins B1, B2, G1and G2Aflatoxin Mig72iB1312 19 300 B2314 20 400 G1328 16 600 G2330 17 900 NOTE A mixture of toluene and aceton

45、itrile (98 g14 2) is used as solvent. 4.14 Stock solution of mixed aflatoxins Prepare a stock solution containing 500 ng/ml of aflatoxin B1, 125 ng/ml of aflatoxin B2, 250 ng/ml of aflatoxin G1and 125 ng/ml of aflatoxin G2in toluene/acetonitrile (4.9). If the solution has to be stored, weigh the fla

46、sk before storage. Wrap the flask tightly in aluminium foil and store it at approximately 4 C. Immediately before use, reweigh the flask and record any change in mass after storage.NOTE Normal exposure to UV light during absorbance measurement results in no observable conversion to photoproducts. 4.

47、15 Standard solution of mixed aflatoxins Transfer each quantity, as specified in Table 2, of mixed aflatoxin stock solution (4.14) into a series of four 2 ml volumetric flasks (5.5). Evaporate the solutions just to dryness under a stream of nitrogen at room temperature. To each flask, add 1 ml of me

48、thanol (4.6). Dissolve the dry residue in it, dilute the solution to the mark with water (4.1) and mix. Prepare the solution freshly on the day of use.6DIN EN ISO 16050:2011-09 EN ISO 16050:2011 (E) Table 2 Preparation of standard solutions Standard solution Volume taken from stock solution Concentration of aflatoxin ng/ml l B1B2G1G21 60 15,0 3,75 7,50 3,75 2 40 10,0 2,50 5,00 2,50 3 20 5,00 1,25 2,50 1,25 4 10 2,50 0,625 1,25 0,625 NOTE The values given are for guidance only. The standard