BS EN 14132-2009 Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up《食品 大麦和焙炒咖啡中赭曲霉素A的测定 免疫亲和柱层纯化的高效液相色谱法(HPLC.pdf

《BS EN 14132-2009 Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up《食品 大麦和焙炒咖啡中赭曲霉素A的测定 免疫亲和柱层纯化的高效液相色谱法(HPLC.pdf》由会员分享，可在线阅读，更多相关《BS EN 14132-2009 Foodstuffs - Determination of ochratoxin A in barley and roasted coffee - HPLC method with immunoaffinity column clean-up《食品 大麦和焙炒咖啡中赭曲霉素A的测定 免疫亲和柱层纯化的高效液相色谱法(HPLC.pdf（18页珍藏版）》请在麦多课文档分享上搜索。

1、BS EN 14132:2009ICS 67.140.20NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Determination ofochratoxin A in barleyand roasted coffee HPLC method withimmunoaffinity columnclean-upThis British Standardwas published under theauthority of the StandardsPo

2、licy and StrategyCommittee on 30 June2009. BSI 2009ISBN 978 0 580 64239 5Amendments/corrigenda issued since publicationDate CommentsBS EN 14132:2009National forewordThis British Standard is the UK implementation of EN 14132:2009. Itsupersedes BS EN 14132:2003 which is withdrawn.The UK participation

3、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 respons

4、ible for its correct application.Compliance with a British Standard cannot confer immunityfrom legal obligations.BS EN 14132:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 14132May 2009ICS 67.140.20 Supersedes EN 14132:2003 English VersionFoodstuffs - Determination of ochratoxin A in barley a

5、nd roastedcoffee - HPLC method with immunoaffinity column clean-upProduits 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 Ochr

6、atoxin A in Gerste undRstkaffee - HPLC-Verfahren mit Reinigung an einerImmunoaffinittssuleThis 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 o

7、f a national standard without any alteration. Up-to-date lists and bibliographical 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 versi

8、on in any other language made by translationunder the responsibility of 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, Denmar

9、k, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEU

10、ROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 14132:2009: EBS EN 14132:2009EN 14132:2009 (E) 2 Contents Foreword 3 1 Scope 4 2 Normative referenc

11、e 4 3 Principle 4 4 Reagents . 4 5 Apparatus . 6 6 Procedure . 7 7 Spiking procedure . 8 8 HPLC analysis 9 9 Calculation 10 10 Precision . 10 11 Test report 11 Annex A (informative) Precision data 12 Bibliography 14 BS EN 14132:2009EN 14132:2009 (E) 3 Foreword This document (EN 14132:2009) has been

12、prepared by Technical Committee CEN/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 November 2009, and conflic

13、ting national standards shall be withdrawn at the latest by November 2009. 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 docume

14、nt will supersede EN 14132:2003 Annex A is informative. WARNING Ochratoxin A is a 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). A

15、cetonitrile is hazardous. Toluene is highly flammable and harmful. Observe appropriate 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 fu

16、me cupboard, such us measurement of standards by UV spectrophotometer, shall be performed 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 Interna

17、l Regulations, the national standards organizations of the following countries are 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

18、, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 14132:2009EN 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 immunoaffini

19、ty column clean up and high performance liquid chromatography (HPLC). This method 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 document

20、s are indispensable 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, Water for analytical laboratory use - Specification and test methods

21、(ISO 3696:1987) 3 Principle Ochratoxin A is extracted from barley by blending 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 i

22、s cleaned up by passing it first through a phenyl silane column and then through 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 g

23、rade and only distilled water or water of grade 1 as defined in EN ISO 3696. 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 phospha

24、te 4.5 Potassium chloride 4.6 Sodium hydroxide solution, (NaOH) = 8,0 g/l Dissolve 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 dihy

25、drogen phosphate (4.4) and 0,2 g of potassium chloride (4.5) in 900 ml of water. 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. BS EN 14132:2009EN 14132:2009 (E)

26、 5 4.8 Sodium hydrogen carbonate solution, (NaHCO3) = 30 g/l In a 1-l-volumetric 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 g

27、lacial acetic acid Mix 99 parts per volume of toluene (4.12) with 1 part 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 o

28、f methanol (4.10) with 1 part per volume of sodium hydrogen carbonate solution (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 pa

29、rts per volume of acetonitrile (4.11) and 2 parts per volume of glacial 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 hydroge

30、n carbonate solution (4.8). 4.19 Phenyl silane column wash reagent 2, (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 par

31、ts per volume of water. 4.21 Ochratoxin A stock solution Dissolve 1 mg 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 determi

32、ne the exact concentration, record the absorption curve between a wavelength 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

33、 per millilitre, using Equation (1): bMAota=100max(1) BS EN 14132:2009EN 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

34、 solvent mixture (4.13), (here: 544 m2/mol); b is the optical path 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

35、mass concentration of ochratoxin A of 10 g/ml. Store this solution 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 mixtu

36、re (4.13). This gives 2 g/ml ochratoxin A solution. Pipette 100 l 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 calibratio

37、n solution containing 20 ng/ml. Prepare the calibration solutions 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). Th

38、is gives a mass concentration of 500 ng/ml ochratoxin A. 4.25 Immunoaffinity 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

39、 solution of 100 ng ochratoxin A in a solvent mixture of the same 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 be

40、d depth and prevent analyte breakthrough). The column should have 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

41、 A. 5 Apparatus Usual laboratory equipment and, in particular, the 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

42、filling them with silanizing reagent and leave this reagent in the vial for 1 min. Rinse the vial twice with appropriate solvent (toluene, acetone or hexane) followed by water (twice) and dry the vial. BS EN 14132:2009EN 14132:2009 (E) 7 5.3 Blender, explosion proof With 1 l capacity jar and cover a

43、nd with a high speed of approximately 20 000 min-1.5.4 Displacement 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, ca

44、pable of pulling a vacuum of 10 mbar and pumping 18 l/min 5.8 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 si

45、ze and 25 mm diameter polysulfone membrane 5.13 HPLC apparatus, 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 separat

46、ing column, for example C18octyldecylsilane (ODS),which ensures 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. 5.1

47、3.4 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 (mesh size = 0,5 mm) bar

48、ley sample into a blender jar (5.3). Add 100 ml of extraction solvent mixture (4.14). Seal the jar and blend for 3 min in a high speed blender at approximately 20 000 min-1. Filter the extract through a filter paper (5.11). 6.1.2 Immunoaffinity column clean-up Prepare the immunoaffinity column accor

49、ding to the suppliers instructions. Pipette 4 ml of the sample filtrate (see 6.1.1) into a 100 ml glass beaker (or similar) and dilute with 44 ml of PBS BS EN 14132:2009EN 14132:2009 (E) 8 (4.7). Connect the immunoaffinity column (4.25) to the vacuum manifold (5.5), and attach the reservoir (5.6) to the immunoaffinity column. Add all the diluted sample extract to the reservoir and pass through the immunoaffinity column. Flow rate should not exceed 5 ml/min. The immunoaffinity column shall not be allowed to run dry. Wash the