EN 15829-2010 en Foodstuffs - Determination of ochratoxin A in currants raisins sultanas mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluor.pdf

1、BS EN 15829:2010ICS 67.050; 67.080.10NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofochratoxin A incurrants, raisins,sultanas, mixed driedfruit and dried figs HPLC methodwith immunoaffinitycolumn cleanup andfluorescence detectionThis

2、British Standardwas published under theauthority of the StandardsPolicy and StrategyCommittee on 28 February2010 BSI 2010ISBN 978 0 580 63017 0Amendments/corrigenda issued since publicationDate CommentsBS EN 15829:2010National forewordThis British Standard is the UK implementation of EN 15829:2010.T

3、he UK participation in its preparation was entrusted to TechnicalCommittee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contrac

4、t. Users are responsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15829:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15829 January 2010 ICS 67.050; 67.080.10 English Version Foodstuffs - Determination of ochratoxin

5、A in currants, raisins, sultanas, mixed dried fruit and dried figs - HPLC method with immunoaffinity column cleanup and fluorescence detection Produits alimentaires - Dosage de lochratoxine A dans les raisins de Corinthe, les raisins secs, les raisins secs de Smyrne, les mlanges de fruits secs et le

6、s figues sches - Mthode CLHP avec purification sur colonne dimmuno-affinit et dtection par fluorescence Lebensmittel - Bestimmung von Ochratoxin A in Korinthen, Rosinen, Sultaninen, gemischtem Trockenobst und getrockneten Feigen - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluores

7、zenzdetektion This European Standard was approved by CEN on 18 December 2009. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibl

8、iographical references concerning such national standards may be obtained on application to the CEN Management Centre 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

9、 of a CEN member into its own language and notified to the CEN Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ic

10、eland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Aven

11、ue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 15829:2010: EBS EN 15829:2010EN 15829:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76

12、Procedure .87 HPLC analysis 98 Calculation . 109 Precision 1110 Test report . 12Annex A (informative) Typical chromatogram 13Annex B (informative) Precision data . 14Bibliography . 15BS EN 15829:2010EN 15829:2010 (E) 3 Foreword This document (EN 15829:2010) has been prepared by Technical Committee C

13、EN/TC 275 “Food analysis Horizontal methods”, the secretariat of which is held by 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 August 2010, and conflicting national standards shall be with

14、drawn at the latest by August 2010. Attention is drawn to the possibility that 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 has been prepared under a mandate gi

15、ve to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic,

16、 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. BS EN 15829:2010EN 15829:2010 (E) 4 1 Scope This Europea

17、n Standard specifies a method for the determination of ochratoxin A in currants, raisins, sultanas, mixed dried fruit and dried figs by high performance liquid chromatography (HPLC) with immunoaffinity cleanup and fluorescence detection. This method has been validated in an interlaboratory study via

18、 the analysis of both naturally contaminated and spiked samples ranging from 1,1 g/kg to 11 g/kg. For further information on the validation, see Clause 9 and Annex B. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address

19、 all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 2 Normative references The following referenced documents are indispens

20、able 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. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:19

21、87) 3 Principle A test portion is extracted with a mixture of methanol and phosphoric acid. The extract is filtered, diluted with phosphate buffered saline, and applied to an immunoaffinity column containing antibodies specific for ochratoxin A. The ochratoxin A is isolated, purified and concentrate

22、d on the column then released with elution solvent. Ochratoxin A is quantified by reverse-phase high performance liquid chromatography (HPLC) with fluorescence detection. 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unl

23、ess otherwise specified. Solvents shall be of quality for HPLC analysis. Commercially available solutions with equivalent properties to those listed may be used. WARNING Dispose of waste solvents according to applicable environmental rules and regulations. Decontamination procedures for laboratory w

24、astes have been reported by the International Agency for Research on Cancer (IARC), see 1. 4.2 Helium purified compressed gas 4.3 Disodium hydrogen phosphate, anhydrous or Na2HPO412 H2O 4.4 Potassium chloride 4.5 Potassium dihydrogen phosphate 4.6 Sodium chloride BS EN 15829:2010EN 15829:2010 (E) 5

25、4.7 Sodium hydroxide 4.8 Ammonium hydroxide solution, substance concentration c(NH4OH) = 1,1 mol/l, for post-column pH shift Prepare fresh when required (optional, see 7.2). 4.9 Hydrochloric acid solution, mass fraction w(HCl) = 37 % in water 4.10 Phosphoric acid solution, c(H3PO4) = 0,1 mol/l 4.11

26、Hydrochloric acid solution, c(HCl) = 0,1 mol/l Dilute 8,28 ml of hydrochloric acid solution (4.9) to 1 l with water. 4.12 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of sodium hydroxide (4.7) in 1 l of water. 4.13 Phosphate buffered saline (PBS) solution Dissolve 8,0 g of sodium chlo

27、ride (4.6), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO412 H2O (4.3), 0,2 g of potassium dihydrogen phosphate (4.5) and 0,2 g of potassium chloride (4.4) in 900 ml of water. After dissolution, adjust the pH to 7,4 with hydrochloric acid solution (4.11) or sodium hydroxide solut

28、ion (4.12) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.14 Acetonitrile WARNING Acetonitrile is hazardous and samples shall be blended using an explosion proof blender which is hous

29、ed within a fume cupboard. After blending, samples shall be filtered inside a fume cupboard. 4.15 Glacial acetic acid, w(CH3COOH) 98 % 4.16 Methanol 4.17 Toluene 4.18 Injection solvent Mix 80 parts per volume of water with 20 parts per volume of acetonitrile (4.14) and two parts per volume of acetic

30、 acid (4.15). 4.19 HPLC mobile phase Mix 99 parts per volume of water with 99 parts per volume of acetonitrile (4.14) and two parts per volume of glacial acetic acid (4.15). Degas the mobile phase solvent with for example helium (4.2). BS EN 15829:2010EN 15829:2010 (E) 6 4.20 Mixture of toluene and

31、glacial acetic acid Mix 99 parts per volume of toluene (4.17) with one part per volume of glacial acetic acid (4.15). 4.21 Immunoaffinity column The immunoaffinity column contains antibodies raised against ochratoxin A. The column shall have a capacity of not less than 100 ng of ochratoxin A and sha

32、ll give a recovery of not less than 70 % when 5 ng of ochratoxin A is applied in a solution of five parts per volume of acetonitrile (4.14) and 95 parts per volume phosphate buffered saline (4.13). 4.22 Surface silanising fluid (optional) Mix one part per volume of the surface silanising fluid with

33、19 parts per volume of toluene (4.17). 4.23 Ochratoxin A, in crystal form or as a film in ampoules 4.24 Ochratoxin A stock solution WARNING Ochratoxin A is a potent nephrotoxin with immunotoxic, teratogenic and potential genotoxic properties. The International Agency for Research on Cancer (IARC) ha

34、s classified ochratoxin A as a possible human carcinogen (group 2B). Protective clothing, gloves and safety glasses should be worn at all times, and all standard and sample preparation stages should be carried out in a fume cupboard. Dissolve 1 mg of the ochratoxin A or the contents of 1 ampoule (if

35、 ochratoxin A has been obtained as a film) in solvent mixture (4.20) to give a solution containing approximately 20 g/ml to 30 g/ml of ochratoxin A. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in a 1 cm quartz cell with solvent mixture

36、(4.20) as reference using the spectrometer (5.12). Identify the wavelength for maximum absorption. Calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre using Equation (1): bMA=100maxota(1) where Amax is the absorption determined at the maximum of the absorption curve (

37、here: at 333 nm); M is the molar mass, in grams per mole, of ochratoxin A (M = 403,8 g/mol); is the molar absorption coefficient, in square metres per mole, of ochratoxin A in the solvent mixture (4.20) (here: 544 m2/mol, see 2); b is the optical path length, in centimetres, of the quartz cell. Stor

38、e this solution in a freezer at approximately - 18 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.25 Ochratoxin A spiking solution Transfer an aliquot of the

39、 stock solution (4.24) containing 12,5 g of ochratoxin A to a 5 ml volumetric flask. Evaporate to dryness under nitrogen at no more than 50 C. Redissolve immediately in methanol (4.16) and make up to volume. This solution contains 2,5 g/ml ochratoxin A. BS EN 15829:2010EN 15829:2010 (E) 7 Store this

40、 solution in a freezer at approximately - 18 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for 12 months. Confirm the concentration of the solution if it is older than six months. 4.26 Ochratoxin A standard solution Transfer 500 l of the ochratoxi

41、n A spiking solution (4.25) to a 5 ml volumetric flask, make up to volume with methanol (4.16). This solution contains 0,25 g/ml ochratoxin A. Store this solution in a freezer at approximately - 18 C. Allow to reach room temperature before opening. A solution stored in this way is usually stable for

42、 12 months. Confirm the concentration of the solution if it is older than six months. 5 Apparatus 5.1 General Usual laboratory glassware and equipment and, in particular the following. 5.2 Silanised glass vials (optional) Prepare the vials by filling them with the silanising reagent (4.22) and leave

43、 this reagent in the vial for 1 min. Rinse the vial first with a solvent of low polarity, for example toluene (4.17) then with methanol (4.16) and dry before use. WARNING The use of silanised glassware may prevent ochratoxin A binding to glass during evaporation. 5.3 High speed blender or homogenize

44、r 5.4 Analytical balance, capable of weighing to 0,000 1 g 5.5 Laboratory balance, capable of weighing to 0,1 g 5.6 Displacement pipettes, adjustable, of 10 ml, 5 ml, 1 ml and 200 l capacity with appropriate pipette tips 5.7 Vacuum manifold, to accommodate immunoaffinity columns 5.8 Reservoirs and a

45、ttachments, to fit to immunoaffinity columns 5.9 Vacuum pump, capable of pulling a vacuum of 1 kPa and pumping 18 l/min 5.10 Filter paper, with pore size of 20 m to 25 m 5.11 HPLC apparatus, comprising the following: 5.11.1 Injection system, capable of injecting e.g. 100 l 5.11.2 Mobile phase pump,

46、isocratic, pulse free, capable of maintaining a volume flow rate of 1 ml/min 5.11.3 Column oven (optional), capable of maintaining a constant temperature above any variability caused by fluctuations in the room temperature (e.g. (45 1) C, 0,5 C temperature repeatability and stability). 5.11.4 Analyt

47、ical reverse-phase HPLC separating column, for example C18octadecylsilane (ODS), length of 25 cm, inner diameter of 4,6 mm and a particle size of 5 m, which ensures resolution of ochratoxin A from BS EN 15829:2010EN 15829:2010 (E) 8 all other peaks. The maximum overlapping of peaks shall be less tha

48、n 10 %. It can be necessary to adjust the mobile phase for a sufficient baseline resolution. A suitable corresponding reverse-phase guard column should be used. 5.11.5 Degasser (optional) 5.11.6 Fluorescence detector, fitted with a flow cell and set at 333 nm (excitation wavelength) and 477 nm (emis

49、sion wavelength), or set at 390 nm (excitation wavelength) and 440 nm (emission wavelength), if an optional post-column system is used. 5.11.7 Recorder, integrator or computer based data processing system 5.11.8 Post-column system (optional), comprising pump, isocratic, pulse free, capable of maintaining a volume flow rate of 0,3 ml/min, zero dead volume t-piece, and reaction coil 1 500 mm 0,25 mm internal diameter tubing (stainless steel or polyetheretherketone (PEEK)