1、September 2009DEUTSCHE NORM English price group 11No part of this standard 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).ICS 67.140.20!$YqY“15478
2、54www.din.deDDIN EN 14132Foodstuffs Determination of ochratoxin A in barley and roasted coffee HPLC method with immunoaffinity column clean-upEnglish version of DIN EN 14132:2009-09Lebensmittel Bestimmung von Ochratoxin A in Gerste und Rstkaffee HPLC-Verfahren mit Reinigung an einer Immunoaffinittss
3、uleEnglische Fassung DIN EN 14132:2009-09SupersedesDIN EN 14132:2003-09 andDIN EN 14132 Corrigendum1:2007-03www.beuth.deDocument comprises pages16DIN EN 14132:2009-09 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat:
4、DIN, Germany). Preliminary work was done by Working Group WG 5 “Biotoxins”. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-03 AA Biotoxin
5、e. The DIN Standard corresponding to the International Standard referred to in this document is as follows: EN ISO 3696 DIN ISO 3696 Amendments This standard differs from DIN EN 14132:2003-09 and DIN EN 14132 Corrigendum 1:2007-03 as follows: a) DIN EN 14132 Corrigendum 1:2007-03 has been incorporat
6、ed, i.e. Equation (1) and the key in subclause 4.21 “Ochratoxin A stock solution” have been corrected. b) The bibliography has been rendered more precise. Previous editions DIN EN 14132: 2003-09 DIN EN 14132 Corrigendum 1: 2007-03 National Annex NA (informative) Bibliography DIN ISO 3696, Water for
7、analytical laboratory use Specification and test methods 2 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14132May 2009ICS 67.140.20 Supersedes EN 14132:2003 English VersionFoodstuffs - Determination of ochratoxin A in barley and roastedcoffee - HPLC method with immunoaffinity column clean-upProd
8、uits alimentaires - Dosage de lochratoxine A danslorge et le caf torrfi - Mthode par purification surcolonne dimmuno-affinit suivie dune analyse parchromatographie liquide haute performance (CLHP)Lebensmittel - Bestimmung von Ochratoxin A in Gerste undRstkaffee - HPLC-Verfahren mit Reinigung an eine
9、rImmunoaffinittssuleThis European Standard was approved by CEN on 24 March 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bib
10、liographical references concerning such nationalstandards 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 translationunder the responsibility o
11、f a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland
12、, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-100
13、0 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14132:2009: EEN 14132:2009 (E) 2 Contents Foreword 3 1 Scope 4 2 Normative reference 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 6 6 Procedure . 7 7 Spiking procedure .
14、 8 8 HPLC analysis 9 9 Calculation 10 10 Precision . 10 11 Test report 11 Annex A (informative) Precision data 12 Bibliography 14 DIN EN 14132:2009-09 EN 14132:2009 (E) 3 Foreword This document (EN 14132:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”,
15、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 November 2009, and conflicting national standards shall be withdrawn at the latest by November 2009. Attent
16、ion 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 will supersede EN 14132:2003 Annex A is informative. WARNING Ochratoxin A is a
17、 potent nephrotoxin and liver toxin and has been reported to have immunosuppressant properties. It is classified by the International Agency for Research on Cancer (IARC) as possibly carcinogenic to humans (Group 2B). Acetonitrile is hazardous. Toluene is highly flammable and harmful. Observe approp
18、riate safety precautions for handling such compounds. Gloves and safety glasses shall be worn at all times and all standard and sample preparation stages shall be carried out in a fume cupboard. Operation outside the fume cupboard, such us measurement of standards by UV spectrophotometer, shall be p
19、erformed with the standard in closed containers. Decontamination procedures for laboratory wastes have been reported by the International Agency for Research on Cancer (IARC), see 1. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries ar
20、e bound to implement this European Standard: Austria, Belgium, Bulgaria, 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, Swed
21、en, Switzerland and the United Kingdom. DIN EN 14132:2009-09 EN 14132:2009 (E) 4 1 Scope This European Standard specifies a method for the determination of ochratoxin A content in barley and roasted coffee using immunoaffinity column clean up and high performance liquid chromatography (HPLC). This m
22、ethod has been validated for ochratoxin A contents in barley in the range from 0,1 g/kg up to 4,5 g/kg and for roasted coffee in the range from 0,2 g/kg up to 5,5 g/kg. 2 Normative reference The following referenced documents are indispensable for the application of this document. For dated referenc
23、es, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. EN ISO 3696, Water for analytical laboratory use - Specification and test methods (ISO 3696:1987) 3 Principle Ochratoxin A is extracted from barley by blendin
24、g with aqueous acetonitrile. The extract is purified by passing it through an immunoaffinity column. Ochratoxin A is extracted from ground roasted coffee by blending with methanol and sodium hydrogen carbonate. The extract is cleaned up by passing it first through a phenyl silane column and then thr
25、ough an immunoaffinity column. Ochratoxin A is separated by reverse-phase HPLC and determined by fluorescence. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or water of grade 1 as defined in EN ISO 3696.
26、 Solvents shall be of quality for HPLC analysis. Commercially available reagents with equivalent properties to the ones listed may be used. 4.2 Sodium chloride 4.3 Disodium hydrogen phosphate 4.4 Potassium dihydrogen phosphate 4.5 Potassium chloride 4.6 Sodium hydroxide solution, (NaOH) = 8,0 g/l Di
27、ssolve 8 g of sodium hydroxide in 900 ml of water, then dilute to 1 l with water. 4.7 Phosphate buffered saline (PBS) Dissolve 8 g of sodium chloride (4.2), 1,2 g of disodium hydrogen phosphate (4.3), 0,2 g of potassium dihydrogen phosphate (4.4) and 0,2 g of potassium chloride (4.5) in 900 ml of wa
28、ter. Adjust the pH to 7,4 with sodium hydroxide solution (4.6) then dilute to 1 l with water. Commercially available phosphate buffered saline tablets with equivalent properties may be used. DIN EN 14132:2009-09 EN 14132:2009 (E) 5 4.8 Sodium hydrogen carbonate solution, (NaHCO3) = 30 g/l In a 1-l-v
29、olumetric flask dissolve 30 g sodium hydrogen carbonate in 900 ml of water. Dilute to volume with water. 4.9 Glacial acetic acid, (CH3COOH) = 98 % 4.10 Methanol 4.11 Acetonitrile 4.12 Toluene 4.13 Solvent mixture of toluene and glacial acetic acid Mix 99 parts per volume of toluene (4.12) with 1 par
30、t per volume of glacial acetic acid (4.9). 4.14 Barley extraction solvent mixture Mix 6 parts per volume acetonitrile (4.11) with 4 parts per volume of water. 4.15 Roasted coffee extraction solvent mixture Mix 1 part per volume of methanol (4.10) with 1 part per volume of sodium hydrogen carbonate s
31、olution (4.8). 4.16 Injection solvent Mix 30 parts per volume of methanol (4.10) with 70 parts per volume of water and 1 part per volume of glacial acetic acid (4.9). 4.17 Mobile phase Mix 102 parts per volume of water with 96 parts per volume of acetonitrile (4.11) and 2 parts per volume of glacial
32、 acetic acid (4.9), filter through a 0,2 m filter (5.12) and degas with for example helium before use. 4.18 Phenyl silane column wash reagent 1 Mix 20 parts per volume of methanol (4.10) with 80 parts per volume of sodium hydrogen carbonate solution (4.8). 4.19 Phenyl silane column wash reagent 2, (
33、NaHCO3) = 1 g/100 ml In a 100 ml volumetric flask dissolve 1 g of sodium hydrogen carbonate in 90 ml water. Dilute to volume with water. 4.20 Phenyl silane column elution reagent Mix 7 parts per volume methanol (4.10) with 93 parts per volume of water. 4.21 Ochratoxin A stock solution Dissolve 1 mg
34、of the ochratoxin A (in crystal form) 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 g/ml to 30 g/ml of ochratoxin A. To determine the exact concentration, record the absorption curve between a wavel
35、ength of 300 nm and 370 nm in 5 nm steps in 1 cm quartz cells (5.14) and solvent mixture (4.13) as reference. Identify the wavelength for maximum absorption and calculate the mass concentration of ochratoxin A, ota, in micrograms per millilitre, using Equation (1): bMAota=100max(1) DIN EN 14132:2009
36、-09 EN 14132:2009 (E) 6 where Amaxis the absorption determined at the maximum of the absorption curve (here: at 333 nm); M is the molar mass of ochratoxin A (M = 403,8 g/mol); is the molar absorption coefficient of ochratoxin A in the solvent mixture (4.13), (here: 544 m2/mol); b is the optical path
37、 length of the quartz cell in centimetres. This solution is stable at 18C for at least 4 years. 4.22 Ochratoxin A standard solution Dilute the stock solution (4.21) with the solvent mixture (4.13) to obtain a standard solution with a mass concentration of ochratoxin A of 10 g/ml. Store this solution
38、 in a refrigerator at approximately 4C and check its stability. 4.23 Ochratoxin A calibration solution Pipette 200 l of the 10 g/ml ochratoxin A standard solution (4.22) into a glass vial and dilute to 1 ml with 800 l of solvent mixture (4.13). This gives 2 g/ml ochratoxin A solution. Pipette 100 l
39、of the 2 g/ml ochratoxin A solution into a glass vial (5.2). Evaporate the solvent under a stream of nitrogen. Redissolve in 10 ml injection solvent (4.16) which has been filtered through a 0,2 m filter (5.12). This gives a calibration solution containing 20 ng/ml. Prepare the calibration solutions
40、at the beginning of every day of the analysis. 4.24 Spiking solution Pipette 100 l of the 10 g/ml ochratoxin A standard solution (4.22) into a glass vial. Dilute to 2 ml with 1,9 ml of the mixture of toluene and acetic acid (4.13). This gives a mass concentration of 500 ng/ml ochratoxin A. 4.25 Immu
41、noaffinity column The immunoaffinity column contains antibodies raised against ochratoxin A. The column shall have a total capacity of not less than 100 ng of ochratoxin A. The performance of the column should be checked by applying a solution of 100 ng ochratoxin A in a solvent mixture of the same
42、composition as the sample extract (6.1.3) to be applied. This shall give a recovery of not less than 85 %. 4.26 Phenyl silane solid phase extraction columns 500 mg sorbent weight and 3 ml reservoir volume (to ensure adequate column bed depth and prevent analyte breakthrough). The column should have
43、a total capacity of not less than 100 ng ochratoxin A and should give a recovery of not less than 85 % when applied in a standard solution of ochratoxin A in the roasted coffee extraction solvent (4.15) containing 100 ng of ochratoxin A. 5 Apparatus Usual laboratory equipment and, in particular, the
44、 following: 5.1 Analytical balance, accurate to 0,01 mg 5.2 Glass vials, of at least 10 ml Certain types of vials might lead to losses of ochratoxin A during evaporation. To avoid this, silanization could be applied. Prepare vials by filling them with silanizing reagent and leave this reagent in the
45、 vial for 1 min. Rinse the vial twice with appropriate solvent (toluene, acetone or hexane) followed by water (twice) and dry the vial. DIN EN 14132:2009-09 EN 14132:2009 (E) 7 5.3 Blender, explosion proof With 1 l capacity jar and cover and with a high speed of approximately 20 000 min-1.5.4 Displa
46、cement pipettes of 5 ml, 1 ml and 200 l capacity with appropriate pipettes tips 5.5 Vacuum manifold to accommodate phenyl silane and immunoaffinity columns 5.6 Reservoirs and attachments to fit to immunoaffinity columns 5.7 Vacuum pump, capable of pulling a vacuum of 10 mbar and pumping 18 l/min 5.8
47、 Wrist action shaker or similar 5.9 Cooling centrifuge capable of 1300 g and operating at 4 C 5.10 Centrifuge tubes, e.g. 50 ml capacity 5.11 Filter paper, pore size 20 m to 25 m or similar 5.12 Disposable syringe filters, of 0,2 m pore size and 25 mm diameter polysulfone membrane 5.13 HPLC apparatu
48、s, consisting of: 5.13.1 Injection system, a syringe-loading injection valve with 100 l injection loop or equivalent 5.13.2 HPLC pump, isocratic, capable of maintaining a volume flow rate of 1 ml/min 5.13.3 Analytical reverse phase separating column, for example C18octyldecylsilane (ODS),which ensur
49、es resolution of ochratoxin A from all other peaks. The maximum overlapping of peaks shall be less than 10 % (it could be necessary to adjust the mobile phase for a sufficient baseline resolution). A suitable pre-column should be used. 5.13.4 Fluorescence detector, fitted with a flow cell and set at 333 nm (excitation) and 460 nm (emission) 5.13.5 Data system 5.14 UV spectrometer, with suitable quartz cells. 6 Procedure 6.1 Barley 6.1.1 Extraction Weigh, to the nearest 0,1 g, a 25 g test portion of the ground (me
