BS EN 14352-2004 Foodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up《食品 基于玉米的食物中fumonisin B1和B2的测定 免疫亲合柱清洁的高效液相.pdf

《BS EN 14352-2004 Foodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up《食品 基于玉米的食物中fumonisin B1和B2的测定 免疫亲合柱清洁的高效液相.pdf》由会员分享，可在线阅读，更多相关《BS EN 14352-2004 Foodstuffs - Determination of fumonisin B1 and B2 in maize based foods - HPLC method with immunoaffinity column clean up《食品 基于玉米的食物中fumonisin B1和B2的测定 免疫亲合柱清洁的高效液相.pdf（22页珍藏版）》请在麦多课文档分享上搜索。

1、BRITISH STANDARD BS EN 14352:2004 Foodstuffs Determination of fumonisin B1 and B2 in maize based foods HPLC method with immunoaffinity column clean up The European Standard EN 14352:2004 has the status of a British Standard ICS 67.060 BS EN 14352:2004 This British Standard was published under the au

2、thority of the Standards Policy and Strategy Committee on 6 August 2004 BSI 6 August 2004 ISBN 0 580 44205 5 National foreword This British Standard is the official English language version of EN 14352:2004. The UK participation in its preparation was entrusted to Technical Committee AW/-/3, Horizon

3、tal analysis, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalog

4、ue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its corre

5、ct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. aid enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor

6、related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 18, an inside back cover and a back cover. The BSI copyright notice displayed in this document indicates when th

7、e document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEANSTANDARD NORMEEUROPENNE EUROPISCHENORM EN14352 July2004 ICS67.060 Englishversion FoodstuffsDeterminationoffumonisinB1andB2inmaize basedfoodsHPLCmethodwithimmunoaffinitycolumnclean up ProduitsalimentairesDos

8、agedesfumonisinesB1etB2 dansdesalimentsbasedemasMthodeCLHPavec purificationparcolonnedimmunoaffinit LebensmittelBestimmungvonFumonisinB1undB2in MaiserzeugnissenHPLCVefahrenmit ImmunoaffinittssulenReinigung ThisEuropeanStandardwasapprovedbyCENon30April2004. CENmembersareboundtocomplywiththeCEN/CENELE

9、CInternalRegulationswhichstipulatetheconditionsforgivingthisEurope an Standardthestatusofanationalstandardwithoutanyalteration.Uptodatelistsandbibliographicalreferencesconcernings uchnational standardsmaybeobtainedonapplicationtotheCentralSecretariatortoanyCENmember. ThisEuropeanStandardexistsinthre

10、eofficialversions(English,French,German).Aversioninanyotherlanguagemadebytra nslation undertheresponsibilityofaCENmemberintoitsownlanguageandnotifiedtotheCentralSecretariathasthesamestatusast heofficial versions. CENmembersarethenationalstandardsbodiesofAustria,Belgium,Cyprus,CzechRepublic,Denmark,E

11、stonia,Finland,France, Germany,Greece,Hungary,Iceland,Ireland,Italy,Latvia,Lithuania,Luxembourg,Malta,Netherlands,Norway,Poland,Portugal, Slovakia, Slovenia,Spain,Sweden,SwitzerlandandUnitedKingdom. EUROPEANCOMMITTEEFORSTANDARDIZATION COMITEUROPENDENORMALISATION EUROPISCHESKOMITEEFRNORMUNG Managemen

12、tCentre:ruedeStassart,36B1050Brussels 2004CEN Allrightsofexploitationinanyformandbyanymeansreserved worldwideforCENnationalMembers. Ref.No.EN14352:2004:EEN 14352:2004 (E) 2 Contents page Foreword3 1 Scope 4 2 Normative references 4 3 Principle4 4 Reagents.4 5 Apparatus .7 6 Sampling.8 7 Procedure .8

13、 8 Calculation10 9 Precision.10 10 Test report 12 Annex A (informative) Precision data14 Annex B (informative) Typical chromatograms16 Bibliography 18 EN 14352:2004 (E) 3 Foreword This document (EN 14352:2004) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”,

14、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 January 2005, and conflicting national standards shall be withdrawn at the latest by January 2005. WARNING

15、Fumonisins are hepatotoxic, nephrotoxic and carcinogenic to rats and mice. Effects on humans are not fully known. Observe appropriate safety precautions for handling fumonisins. Any laboratory spills should be washed with 5 % solution of sodium hypchlorite. Acetonitrile is hazardous and samples shal

16、l be shaken using a shaker, which is housed within a fume cupboard. 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

17、establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium

18、, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN 14352:2004 (E) 4 1 Scope This document sp

19、ecifies a method for the detemination of fumonisin B 1(FB 1 ) and fumonisin B 2(FB 2 ) in maize based foods using high performance liquid chromatography (HPLC) and immunoaffinity clean-up, see 1, 2, 3. The method has been successfully validated in a collaborative study according to AOAC Guidelines f

20、or collaborative study procedures 4 to validate characteristics of a method of analysis for the determination of fumonisins in maize flour and corn flakes containing 323 g/kg to 1414 g/kg FB 1and 90 g/kg to 558 g/kg FB 2 . 2 Normative references The following referenced documents are indispensable f

21、or 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 (ISO 3696:1987). 3 Pr

22、inciple Fumonisins are extracted from the sample with a mixture of water, methanol and acetonitrile. The filtered extract is purified by immunoaffinity column and fumonisins are eluted with methanol. The extract is evaporated and the residue is redissolved in a mixture of acetonitrile and water and

23、o-phthaldialdehyde-2- mercaptoethanol (OPA-MCE) is added to form fluorescent fumonisin derivatives. The derivatives are analysed by reverse-phase high performance liquid chromatography (RP-HPLC) with fluorescence detection. 4 Reagents 4.1 General During the analysis, unless otherwise stated, use onl

24、y reagents of recognized analytical grade and only distilled water or water of grade 1 according to EN ISO 3696. Solvents shall be of quality for HPLC analysis. 4.2 Methanol 4.3 Acetonitrile 4.4 o-phosphoric acid, volume fraction (H 3 PO 4 ) = 85 % 4.5 o-phthaldialdehyde (OPA) 4.6 2-mercaptoethanol

25、(MCE) 4.7 Sodium dihydrogen phosphate solution, substance concentration c(NaH 2 PO 4 2H 2 O) = 0,1 mol/l Dissolve 15,6 g of NaH 2 PO 4 2H 2 O in 1 l of distilled water. EN 14352:2004 (E) 5 4.8 Disodium tetraborate solution, c(Na 2 B 4 O 7 10H 2 O) = 0,1 mol/l Dissolve 3,8 g Na 2 B 4 O 7 10H 2 O in 1

26、00 ml of distilled water. 4.9 Sodium chloride (NaCl) 4.10 Disodium hydrogen phosphate (Na 2 HPO 4 ) 4.11 Potassium dihydrogen phosphate (KH 2 PO 4 ) 4.12 Potassium chloride (KCl) 4.13 Hydrochloric acid (HCl), concentrated 4.14 Extraction solvent Mix 25 volume parts of acetonitrile (4.3) with 25 volu

27、me parts of methanol (4.2) and 50 volume parts of water. 4.15 Acetonitrile-water solution, volume fraction (CH 3 CN) = 50 % Mix 50 volume parts of acetonitrile (4.3) with 50 volume parts of water. 4.16 Phosphate buffered saline (PBS) Dissolve 8,0 g of sodium chloride (4.9), 1,2 g of disodium hydroge

28、n phosphate (4.10), 0,2 g of potassium dihydrogen phosphate (4.11) and 0,2 g of potassium chloride (4.12) in approximately 990 ml of water. Adjust pH to 7,0 with concentrated hydrochloric acid (4.13) and bring to 1 l with water. Phosphate buffered saline tablet or ready-to-use solutions can also be

29、used. 4.17 Immunoaffinity column (IAC) The column shall contain antibodies raised against FB 1and fumonisin FB 2 . The column shall have a total capacity of not less than 5 g of fumonisins and shall give a recovery of not less than 90 % for FB 1and FB 2when applied as a standard solution in a mixtur

30、e of methanol and PBS containing 5 g of fumonisins. Up to 10 volume parts of methanol or acetonitrile may be used in the mixture with PBS. The columns shall be warmed up to room temperature before use. 4.18 HPLC mobile phase Mix 77 volume parts of methanol (4.2) with 23 volume parts of sodium dihydr

31、ogen phosphate solution (4.7). Adjust to pH 3,35 with o-phosphoric acid (4.4). Filter the solution through a membrane filter (5.14). The mobile phase composition may have to be adjusted to conform to individual HPLC column characteristics. 4.19 Derivatization reagent Dissolve 40 mg of OPA (4.5) in 1

32、 ml of methanol (4.2) and dilute with 5 ml of disodium tetraborate solution (4.8). Add 50 l of MCE (4.6) and mix. The solution is stable for up to one week at room temperature in the dark in a capped amber vial. 4.20 Stock solutions of FB 1and FB 2Prepare a stock solution of FB 1and a stock solution

33、 of FB 2in acetonitrile-water (4.15) at a mass concentration of 50 g/ml for each standard substance. Store the solutions at approximately 4 C. EN 14352:2004 (E) 6 Fumonisin stock solutions are stable for at least 6 months when stored at approximately 4 C. 4.21 Mixed fumonisins stock solution Prepare

34、 a mixed stock solution by pipetting 1000 l of the FB 1stock solution and 500 l of the FB 2stock solution into a 5 ml volumetric flask. Dilute to the mark with the acetonitrile-water solution (4.15) and shake well to obtain a mixed stock solution containing 10 ng FB 1 /l and 5 ng FB 2 /l. This solut

35、ion is stable at + 4 C for at least 6 months. Smaller volumes may be used to prepare the mixed fumonisins stock solution. 4.22 Mixed fumonisins standard solutions for HPLC Prepare four HPLC mixed standard solutions in 5 ml volumetric flasks according to Table 1. Dilute each standard solution to volu

36、me (5 ml) with acetonitrile-water solution (4.15) and mix well. This solution is stable at + 4 C for at least 6 months. Smaller volumes may be used to prepare the mixed fumonisins standard solution. Table 1 Preparation of mixed standard solutions for HPLC Final fumonisin concentration of mixed stand

37、ard solution, ng/l Mixed standard solution Volume taken from mixed stock solution (l) Addition of acetonitrile- water solution (l) FB 1FB 21 50 4 950 0,10 0,050 2 125 4 875 0,25 0,125 3 500 4 500 1,00 0,500 4 1 000 4 000 2,00 1,000 EN 14352:2004 (E) 7 5 Apparatus 5.1 Usual laboratory equipment and,

38、in particular, the following: 5.2 Orbital shaker 5.3 Centrifuge bottle of 250 ml capacity with screw cap 5.4 Centrifuge capable of a centrifugal force up to 2 500 g 5.5 Filter papers, with a pore size of 20 m to 25 m 5.6 Glass microfiber filter papers, with a pore size of 11 m 5.7 Reservoir, 25 ml w

39、ith luer tip connector for immunoaffinity column (IAC) 5.8 Microlitre syringe(s) or calibrated micropipette(s), 25 l to 1 000 l 5.9 Laboratory balance, capable of weighing to the nearest 0,01 g 5.10 Analytical balance capable of weighing to the nearest 0,1 mg 5.11 Vacuum manifold to accommodate immu

40、noaffinity columns 5.12 Vortex mixer 5.13 Solvent evaporator, with heating module, or similar. 5.14 Membrane filter for aqueous solutions, with a pore size of 0,45 m. 5.15 HPLC apparatus, comprising the following 5.15.1 HPLC pump, isocratic, suitable for e.g. 1 ml/min constant flow rate 5.15.2 Injec

41、tion system capable to deliver e.g. 20 l 5.15.3 Analytical reverse-phase separating column, for example octyldecylsilane (ODS), which ensures a baseline resolution of the fumonisin peaks from all other peaks, with the following characteristics: stainless steel; a length of 150 mm; an inner diameter

42、of 4,6 mm; a stationary phase with particle size of 5 m; a suitable corresponding reverse-phase guard column. EN 14352:2004 (E) 8 Columns of other dimensions may also be used. 5.15.4 Fluorescence detector, fitted with a flow cell and set at 335 nm (excitation) and 440 nm (emission). Detection of at

43、least 0,5 ng of FB 1and FB 2should be possible (signal to noise = 3). 5.15.5 Data system 6 Sampling It is important that the laboratory receives a sample, which is truly representative and has not been damaged during transport or storage. 7 Procedure 7.1 Preparation of the test sample Grind the samp

44、le to pass through a 1 mm sieve and homogenize the sample. 7.2 Extraction Weigh, to the nearest 0,1 g, a 20 g test sample into a 250 ml centrifuge bottle (5.3) and add 50 ml of extraction solvent (4.14). Cover centrifuge bottle and shake for 20 min with orbital shaker (5.2). Centrifuge (5.4) for 10

45、min at 2 500 g and filter the supernatant through filter paper (5.5) avoiding transferring solid material on the filter. Extract again the remaining solid material by adding 50 ml of extraction solvent to the centrifuge bottle and shake again for 20 min. Centrifuge for 10 min at 2500 g and filter th

46、e extract through the filter paper. Mix the two extract filtrates and pipette 10 ml of filtrate into 100 ml flask. Add 40 ml of PBS (4.16) to the 10 ml filtrate and mix well. Filter the diluted extract through microfiber filter (5.6) and collect 10 ml of filtrate (equivalent to 0,4 g test sample) th

47、at will be cleaned-up through immunoaffinity column (4.17). 7.3 Immunoaffinity clean-up Remove top cap from column and connect with reservoir (5.7). Remove end cap from column and attach to vacuum manifold (5.11). Pipet 10 ml aliquot portion of the filtrated sample extract into reservoir. Let filtra

48、te flow through column at approximately 1 to 2 drops per second and discard the eluate. Wash column with 10 ml PBS (4.16) at a rate of 1 to 2 drops per second until air comes through the column. Discard washing and place a 4 ml vial under column. Elute fumonisins with 1,5 ml methanol (4.2), at a flow rate not more than 1 drop per second. Evaporate (5.13) the eluate to dryness under a stream of nitrogen at approximately 60 C or less. Retain the dried residue at approximately 4 C until derivatization