1、BRITISH STANDARDBS ISO 14674:2005Milk and milkpowder Determination of aflatoxin M1content Clean-up by immunoaffinitychromatography and determination bythin-layerchromatographyICS 67.100.10g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51
2、g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS ISO 14674:2005This British Standard was published under the authority of the Standards Policy and Strategy Committee on 1 August 2005 BSI 1 August 2005ISBN 0 580 46228 5National forewordThis British Standard r
3、eproduces verbatim ISO 14674:2005 and implements it as the UK national standard.The UK participation in its preparation was entrusted to Technical Committee AW/5, Milk and milk products, which has the responsibility to: aid enquirers to understand the text; present to the responsible international/E
4、uropean committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; monitor related international and European developments and promulgate them in the UK.A list of organizations represented on this committee can be obtained on request to its secretary.Cros
5、s-referencesThe British Standards which implement international publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Stand
6、ards Online.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations.Summary of pagesThis document comprises a front cover,
7、an inside front cover, the ISO title page, pages ii to v, a blank page, pages 1 to 11 and a back cover.The BSI copyright notice displayed in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsReference numbersISO 14674:2005(E)IDF 190:20
8、05(E)INTERNATIONAL STANDARD ISO14674IDF190First edition2005-05-01Milk and milk powder Determination of aflatoxin M1content Clean-up by immunoaffinity chromatography and determination by thin-layer chromatography Lait et lait en poudre Dtermination de la teneur en aflatoxin M1 Purification par chroma
9、tographie dimmunoaffinit et dtermination par chromatographie sur couche mince BS ISO 14674:2005 iiiiiContents PageForeword iv1 Scope 12 Terms and definitions. 13 Principle . 14 Reagents 25 Apparatus. 36 Sampling 47 Preparation of test samples. 57.1 Milk or liquid milk products . 57.2 Milk powders .
10、57.3 Immunoaffinity clean-up. 58 Procedure. 68.1 Unidirectional TLC 68.2 Bidirectional TLC 69 Calculation and expression of results 79.1 Calculation. 79.2 Expression of results 810 Precision 810.1 Interlaboratory test . 810.2 Repeatability 810.3 Reproducibility 811 Test report 9Annex A (informative)
11、 Results of interlaboratory test 10Bibliography . 11BS ISO 14674:2005 ivForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO tech
12、nical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
13、 with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft I
14、nternational Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document
15、 may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 14674IDF 190 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaborat
16、ion with AOAC International. It is being published jointly by ISO and IDF and separately by AOAC International. BS ISO 14674:2005vForeword IDF (the International Dairy Federation) is a worldwide federation of the dairy sector with a National Committee in every member country. Every National Committe
17、e has the right to be represented on the IDF Standing Committees carrying out the technical work. IDF collaborates with ISO and AOAC International in the development of standard methods of analysis and sampling for milk and milk products. Draft International Standards adopted by the Action Teams and
18、 Standing Committees are circulated to the National Committees for voting. Publication as an International Standard requires approval by at least 50 % of the National Committees casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of pat
19、ent rights. IDF shall not be held responsible for identifying any or all such patent rights. ISO 14674IDF 190 was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and milk products, and the International Dairy Federation (IDF), in collaboration with AOAC Internationa
20、l. It is being published jointly by ISO and IDF and separately by AOAC International. All work was carried out by the Joint ISO/IDF/AOAC Action Team, Organic contaminants, of the Standing Committee on Analytical methods for additives and contaminants, under the aegis of its project leader, Mrs S Dra
21、gacci (FR). BS ISO 14674:2005 blank1Milk and milk powder Determination of aflatoxin M1content Clean-up by immunoaffinity chromatography and determination by thin-layer chromatography WARNING The method described in this International Standard requires the use of aflatoxin M1solutions. Aflatoxins are
22、 carcinogenic to human subjects. Attention is drawn to the statement made by the International Agency for Research on Cancer (WHO).5Aflatoxins are subject to light degradation. Adequately protect analytical work from daylight and keep aflatoxin standard solutions protected from light, for example by
23、 using amber vials or aluminium foil. The use of non-acid-washed glassware (e.g. vials, tubes, flasks) for aqueous aflatoxin solutions can cause a loss of aflatoxin. Take special care with new glassware. Before use, soak the new glassware in diluted acid (e.g. 2 mol/l sulfuric acid) for several hour
24、s, then rinse extensively with distilled water to remove all traces of acid. Check to ensure that the pH is in the range of 6 to 8 by using a pH-paper. Use the decontamination procedure for laboratory wastes developed and validated by the International Agency for Research on Cancer (WHO).51 Scope Th
25、is International Standard specifies a method for the determination of the aflatoxin M1(AFM1) content of milk and milk powder by a method including a clean-up step using immunoaffinity chromatography followed by a thin-layer chromatography (IAC-TLC). The method is applicable to raw milk, low fat or s
26、kimmed liquid milk and milk powder. The lowest quantity of AFM1 that can commonly be determined is 2 ng, which corresponds to a limit of quantification close to 0,10 g/l for liquid milk or dissolved milk powder (for a spot of 20 l). 2 Terms and definitions For the purposes of this document, the foll
27、owing terms and definitions apply. 2.1aflatoxin M1content mass fraction of substances determined by the method specified in this International Standard NOTE The aflatoxin M1content is expressed as micrograms per litre for liquid milk products, and as micrograms per kilogram for milk powder. 3 Princi
28、ple Aflatoxin M1(AFM1) is extracted by passing the test portion through an immunoaffinity column. The column contains specific antibodies bound onto a solid support material. As the sample passes through the column, the antibodies selectively bind with any AFM1 (antigen) present and form an antibody
29、-antigen complex. All BS ISO 14674:2005 2other components of the sample matrix are washed off the column with water. Then the AFM1 is eluted from the column with methanol and acetonitrile. After concentration of the eluate, the amount of AFM1 is determined by one-dimensional thin-layer chromatograph
30、y. In the case of interference, two-dimensional thin-layer chromatography is carried out to separate the AFM1 from its impurities. 4 Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. 4.1 Pure sol
31、vents.WARNING Some of the pure solvents (e.g. chloroform, acetonitrile, toluene and methanol) are toxic. Take all necessary precautions where needed. 4.1.1 Chloroform (CHCl3).4.1.2 Acetonitrile (CH3CN).4.1.3 Diethyl ether (C2H5)2O. 4.1.4 Methanol (CH3OH). 4.1.5 Toluene (C6H5CH3).4.1.6 Acetone (CH3.C
32、OCH3), optional. 4.1.7 Isopropanol (CH3CHOHCH3), optional. 4.2 Acetonitrile/methanol solution, of volume ratio 3:2. Add 30 ml of acetonitrile (4.1.2) to 20 ml of methanol (4.1.4) and mix. 4.3 Toluene/acetonitrile solution, of volume ratio 9:1. Add 9 ml of toluene (4.1.5) to 1 ml of acetonitrile (4.1
33、.2) and mix. Use this solution to resuspend the AFM1 standard solutions (4.5) and the evaporated eluate before the TLC analysis. 4.4 TLC development solvents.4.4.1 Unidirectional TLC solution.Prepare a 100 ml unidirectional TLC solution by adding 4 ml of methanol (4.1.4) and 1 ml of water to 95 ml o
34、f diethyl ether (4.1.3) and mix well (volume ratio 95:4:1). 4.4.2 Bidirectional TLC solution, optional. Prepare a 100 ml bidirectional TLC solution by adding 10 ml of acetone (4.1.6) and 3 ml of isopropanol (4.1.7) to 87 ml of chloroform (4.1.1) and mix well (volume ratio 87:10:3). 4.5 Aflatoxin M1s
35、tandard solution.4.5.1 AFM1 standard stock solution.Prepare an AFM1 standard stock solution with a nominal concentration of 10 g/ml chloroform (4.1.1); i.e. by resuspending a lyophilized film of 10 g of AFM1 to 1 ml of chloroform. BS ISO 14674:20053In accordance with the AOAC protocol 6, determine t
36、he concentration of the AFM1 standard stock solution by measuring its absorbance at the wavelength of maximum absorption and use a calibrated spectrometer to record the absorbance of the standard stock solution against chloroform (4.1.1), used as blank, at between O 200 nm and O 400 nm. Check the pu
37、rity of the AFM1 by recording the spectrum between 200 nm and 400 nm. Measure the absorbance (A) at the wavelength for maximum absorption (Omax), i.e. close to 365 nm. Calculate the mass concentration, c, expressed in micrograms per millilitre, by using the following equation: 100 /cAM H uuwhere A i
38、s the numerical value of the absorbance measured at Omax;M is the numerical value of the molar mass of the AFM1, in grams per mole (M 328 g/mol); H is the numerical value of the absorption coefficient of AFM1 in chloroform, in square metres per mole (H 1 995 m2mol1).Keep the AFM1 standard stock solu
39、tion in a well-stoppered amber-coloured vial protected from light. Store the standard solution at below 0 C. Under these conditions, the AFM1 standard stock solution is stable for about one year. 4.5.2 AFM1 standard working solution.4.5.2.1 Working solution A.Use a volumetric pipette or a Hamilton-l
40、ike microsyringe (5.2) to transfer 50 l of AFM1 standard stock solution (4.5.1) into a vial. Evaporate the solution to dryness. Resuspend the dried solution with 500 l of toluene/acetonitrile solution (4.3) to obtain an AFM1 standard working solution with concentration of 1 g/ml (working solution A)
41、. Use solution A to spot onto TLC plates for test samples with a high contamination level or when the determination level is close to 0,50 g/l. 4.5.2.2 Working solution B.Transfer 100 l of solution A to a vial. Add 900 l of toluene/acetonitrile solution (4.3) to obtain an AFM1 standard working solut
42、ion with concentration of 0,1 g/ml (working solution B). Use solution B to spot onto TLC plates for test samples with a low contamination level or when the determination level is close to 0,10 g/l. 5 Apparatus Usual laboratory apparatus and, in particular, the following. 5.1 Volumetric pipettes, of
43、required capacities. 5.2 Hamilton-like microsyringes.1)5.3 Laboratory glassware, such as glass beakers and funnels, of appropriate capacities. 1) Hamilton-like syringes and Whatman No. 4 are examples of suitable products available commercially. This information is given for the convenience of users
44、of this document and does not constitute an endorsement by either ISO or IDF of these products. BS ISO 14674:2005 BS ISO 14674:200545.4 One-mark volumetric flask, of capacity 200 ml. 5.5 Measuring cylinder, of capacity 100 ml. 5.6 Disposable syringes, of capacities 10 ml and 100 ml. 5.7 Glass conica
45、l tube, of capacity 5 ml, to collect the extract after the clean-up step. 5.8 Evaporation system, rotary evaporator or by using a gentle stream of nitrogen. 5.9 Spectrometer, capable of measuring at wavelengths at between O 200 nm and O 400 nm, with quartz face cells of optical length 1 cm. 5.10 Wat
46、er bath, capable of operating at between 35 C and 37 C. 5.11 Centrifuge, capable of producing a radial acceleration of at least 2 000g and, if possible, capable of cooling to 4 C. 5.12 Filter paper (Whatman No. 41)or equivalent). 5.13 Affinity columns The immunoaffinity columns shall contain antibod
47、ies against AFM1. The columns shall have a maximum capacity of not less than 100 ng of AFM1 (which corresponds to 1 g/l or 10 g/kg when 100 ml of test portion is applied). They shall give a recovery of not less than 80 % for AFM1 when a calibrant solution containing 4 ng of toxin is applied (corresp
48、onding to 0,04 g/l or 0,40 g/kg applying a 100 ml of test portion). Any immunoaffinity column meeting the above-mentioned performance specifications may be used. 5.14 Analytical balance, capable of weighing to the nearest 0,1 g. 5.15 Magnetic stirrer.5.16 Vortex type stirrer.5.17 Vacuum system, for
49、immunoaffinity cartridge. 5.18 Oven, capable of maintaining a temperature of approx. 50 C. 5.19 Thin-layer chromatography (TLC) container.5.20 Thin-layer chromatography (TLC) plates, of surface 10 cm u 10 cm or 20 cm u 20 cm, with silica gel 60, made of glass or aluminium. 5.21 UV lamp, capable of operating at 365 nm. 5.22 Densitometer (optional). 6 SamplingA representative sample should have been sent to the laboratory. It should not have been damaged or changed during transport or storage. Sampling
