1、BS EN 15835:2010ICS 67.050; 67.230NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofochratoxin A in cerealbased foods for infantsand young children HPLC methodwith immunoaffinitycolumn cleanup andfluorescence detectionThis British Standa
2、rdwas published under theauthority of the StandardsPolicy and StrategyCommittee on 28 February2010 BSI 2010ISBN 978 0 580 63016 3Amendments/corrigenda issued since publicationDate CommentsBS EN 15835:2010National forewordThis British Standard is the UK implementation of EN 15835:2010.The UK particip
3、ation 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 contract. Users are r
4、esponsible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 15835:2010EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 15835 February 2010 ICS 67.050; 67.230 English Version Foodstuffs - Determination of ochratoxin A in cereal base
5、d foods for infants and young children - HPLC method with immunoaffinity column cleanup and fluorescence detection Produits alimentaires - Dosage de lochratoxine A dans les aliments base de crales pour nourrissons et jeunes enfants - Mthode CLHP avec purification sur colonne dimmuno-affinit et dtect
6、ion par fluorescence Lebensmittel - Bestimmung von Ochratoxin A in Suglings-und Kleinkindernahrung auf Getreidebasis - HPLC-Verfahren mit Reinigung an einer Immunoaffinittssule und Fluoreszenzdetektion This European Standard was approved by CEN on 25 December 2009. CEN members are bound to comply wi
7、th 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 bibliographical references concerning such national standards may be obtained on application to the CEN Management C
8、entre 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 notified to the CEN Management Centre has the same status as the offi
9、cial versions. 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, Slo
10、vakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2010 CEN All rights of exploitation in any form and by any means reserved worldwid
11、e for CEN national Members. Ref. No. EN 15835:2010: EBS EN 15835:2010EN 15835:2010 (E) 2 Contents Page Foreword 31 Scope 42 Normative references 43 Principle 44 Reagents .45 Apparatus .76 Procedure .97 HPLC analysis . 108 Calculation . 119 Precision 1210 Test report . 13Annex A (informative) Typical
12、 chromatograms 14Annex B (informative) Precision data . 15Annex C (informative) Data on the in-house study . 16Bibliography . 18BS EN 15835:2010EN 15835:2010 (E) 3 Foreword This document (EN 15835:2010) has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods”, the secre
13、tariat 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 withdrawn at the latest by August 2010. Attention is drawn
14、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 given to CEN by the European Commission and the European
15、Free Trade Association. Annexes A, B and C are informative. 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, Denmark, Estonia,
16、 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 15835:2010EN 15835:2010 (E) 4 1 Scope This European Standard specifi
17、es a method for the determination of ochratoxin A in cereal based foods for infants and young children by high performance liquid chromatography (HPLC) with immunoaffinity column cleanup and fluorescence detection. This method has been validated in an interlaboratory study via the analysis of both n
18、aturally contaminated and spiked samples ranging from 0,050 g/kg to 0,217 g/kg. For further information on the validation see Clause 8 and Annex B. Additional studies have shown that this method is applicable to cereal based baby foods containing 8 different types of cereals, honey and cocoa, at lev
19、els up to 3,540 g/kg, see Annex C and 6. WARNING The use of this standard can involve hazardous materials, operations and equipment. This standard does not purport to address all the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropria
20、te safety and health practices and determine the applicability of regulatory limitations prior to use. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the l
21、atest edition of the referenced document (including any amendments) applies. EN ISO 3696:1995, Water for analytical laboratory use Specification and test methods (ISO 3696:1987) 3 Principle A test portion is extracted with tert-butyl methyl ether after addition of 0,5 mol/l phosphoric acid / 2 mol/l
22、 sodium chloride solution. The extract is evaporated and redissolved in methanol and phosphate buffered saline (PBS) solution. After removal of lypophilic compounds with hexane, the extract is applied to an immunoaffinity column containing antibodies specific to ochratoxin A. The toxin is eluted fro
23、m the column with methanol. Ochratoxin A is determined by HPLC with enhanced fluorescence detection involving post column reaction with ammonia. NOTE Some investigations indicate that HPLC can be also performed without the use of ammonia although this results in at least a two-fold decrease of the r
24、esponse for ochratoxin A. In this case, the fluorescence detection conditions need to be changed (excitation wavelength = 333 nm, emission wavelength = 460 nm). 4 Reagents 4.1 General Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696:1995, unless otherw
25、ise 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 wastes have
26、 been reported by the International Agency for Research on Cancer (IARC), see 4. 4.2 Helium purified compressed gas 4.3 Nitrogen BS EN 15835:2010EN 15835:2010 (E) 5 4.4 Disodium hydrogen phosphate, Na2HPO4 anhydrous or Na2HPO412 H2O 4.5 Potassium chloride 4.6 Potassium dihydrogen phosphate 4.7 Sodiu
27、m chloride 4.8 Sodium hydroxide 4.9 Ammonium hydroxide solution, the mass fraction w(NH4OH) = 25 % in water (post column reagent) Degas the solution with a degasser (5.21.7). 4.10 Hydrochloric acid solution, w(HCl) is 37 % (acidimetric) 4.11 Phosphoric acid solution, w(H3PO4) = 85 % 4.12 Hydrochlori
28、c acid solution, c(HCl) = 0,1 mol/l Dilute 8,28 ml of hydrochloric acid solution (4.10) to 1 l of water. 4.13 Sodium hydroxide solution, c(NaOH) = 0,1 mol/l Dissolve 4 g of sodium hydroxide (4.8) in 1 l of water. 4.14 Phosphate buffered saline (PBS) solution, c(NaCl) = 120 mmol/l, c(KCl) = 2,7 mmol/
29、l, c(phosphate buffer) = 10 mmol/l, pH = 7,4 Dissolve 8,0 g of sodium chloride (4.7), 1,2 g of anhydrous disodium hydrogen phosphate or 2,9 g of Na2HPO412 H2O (4.4), 0,2 g of potassium dihydrogen phosphate (4.6) and 0,2 g of potassium chloride (4.5) in 900 ml of water. After dissolution, adjust the
30、pH to 7,4 with hydrochloric acid solution (4.12) or sodium hydroxide solution (4.13) as appropriate, then dilute to 1 l with water. Alternatively, a PBS solution with equivalent properties can be prepared from commercially available PBS material. 4.15 Mixture of phosphoric acid solution and sodium c
31、hloride solution, c(H3PO4) = 0,5 mol/l, c(NaCl) = 2 mol/l Dissolve 118 g of sodium chloride (4.7) in approximately 900 ml of water. Add 33 ml of phosphoric acid (4.11) and make up to 1,0 l with water. 4.16 Glacial acetic acid, the mass fraction 99,7 % 4.17 Acetic acid solution, the volume fraction i
32、s 9 % Add 90 ml of glacial acetic acid (4.16) and 910 ml of water. 4.18 Hexane WARNING Hexane is highly flammable. Operations involving this solvent shall be performed in a fume cupboard. Serious health problems can be derived from prolonged exposure to this reagent. BS EN 15835:2010EN 15835:2010 (E
33、) 6 4.19 Methanol, gradient grade 4.20 Toluene 4.21 Mixture of methanol and acetic acid solution Mix 72 parts per volume of methanol (4.19) with 28 parts per volume of acetic acid solution (4.17). 4.22 Tert-butyl methyl ether WARNING Tert-butyl methyl ether is hazardous and samples shall be blended
34、using an explosion proof blender which is housed within a fume cupboard. Centrifugation of extracts shall be performed at cool temperature (4 C to 8 C). 4.23 Mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.20) with one part per volume of glacial acetic acid (4.16). 4
35、.24 HPLC mobile phase A Acetic acid solution (4.17). 4.25 HPLC mobile phase B Methanol (4.19). Degas the mobile phases A and B with for example helium (4.2). Helium can be pumped into the reservoirs of both mobile phases A and B. The pumping rate should be 50 ml/min to 100 ml/min. The use of a degas
36、ser is also an acceptable option. 4.26 Immunoaffinity columns The immunoaffinity column contain antibodies raised against ochratoxin A. The column shall have a capacity of not less than 100 ng of ochratoxin A and shall give a recovery of not less than 85 % when applied as a standard solution of ochr
37、atoxin A in a mixture of 15 parts per volume of methanol (4.19) and 85 parts per volume of PBS solution (4.14) containing 3 ng of ochratoxin A. 4.27 Ochratoxin A, in crystal form or as a film in ampoules WARNING Ochratoxin A is a potent nephrotoxin with immunotoxic, teratogenic and potential genotox
38、ic properties. The International Agency for Research on Cancer (IARC) has 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 cupb
39、oard. 4.28 Ochratoxin A stock solution Prepare a stock solution of ochratoxin A in the mixture of toluene and glacial acetic acid (4.23) with a nominal concentration of 10 g/ml. To determine the exact concentration, record the absorption curve between a wavelength of 300 nm and 370 nm in 5 nm steps
40、in 1 cm quartz cells (5.22) in a spectrometer with the solvent mixture (4.23) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, using Equation (1): bMA=100maxota(1) BS EN 15835:2010EN 15835:2010 (E) 7
41、 where Amax is the absorption determined at the maximum of the absorption curve (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 (4.23), (here: 544 m2/m
42、ol); b is the path length, in centimetres, of the quartz cell. 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 12 months. Confirm the concentration of the solution if it is older than six mo
43、nths. 4.29 Ochratoxin A standard solution Pipette a volume of ochratoxin A stock solution (4.28) containing exactly 400 ng ochratoxin A into a 10 ml calibrated volumetric flask (5.13) and dilute to 10 ml with the mixture of toluene and glacial acetic acid (4.23) and shake. This gives a standard solu
44、tion containing 40,0 ng/ml of 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 12 months. Confirm the concentration of the solution if it is older than six months. 4.30 Ochratox
45、in A spiking solution Pipette a volume of ochratoxin A stock solution (4.28) containing exactly 2 500 ng ochratoxin A into a 50 ml calibrated volumetric flask (5.13) and dilute to 50 ml with the mixture of toluene and glacial acetic acid (4.23) and shake. This gives a spiking solution containing 50,
46、0 ng/ml of 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 12 months. Confirm the concentration of the solution if it is older than six months. 5 Apparatus 5.1 General Usual la
47、boratory glassware and equipment and, in particular, the following: 5.2 High speed blender 5.3 Analytical balance, capable of weighing to 0,000 1 g 5.4 Laboratory balance, capable of weighing to 0,1 g 5.5 Vacuum manifold, to accommodate immunoaffinity columns 5.6 Filter papers, suitable for qualitat
48、ive analysis 5.7 pH indicator paper, for pH = 0 to pH = 14 5.8 Cooling centrifuge, capable of a centrifugal force of 15 300 g at 4 C BS EN 15835:2010EN 15835:2010 (E) 8 5.9 Centrifuge bottles, of 250 ml capacity with screw cap, chemically resistant to tert-butyl methyl ether and able to work at 15 3
49、00 g without deformation 5.10 Rotary evaporator, with water bath 5.11 Disposable syringe barrels, to be used as reservoirs, of 5 ml, 20 ml and 50 ml capacity, luer locks and attachments to fit to immunoaffinity columns 5.12 Microsyringes, of 25 l, 50 l, 100 l, 500 l and 1 000 l capacity 5.13 Calibrated volumetric flasks, e.g. of 10 ml, 50 ml and 1 000 ml capacity 5.14 Vacuum system 5.15 Round bottomed flasks, of 100 ml capacity 5.16 Calibrated volumetric pipettes 5.17 Displac
