【商检行业标准(SN)】 进出口粮谷中玉米赤霉烯酮的测定 免疫亲合柱-液相色谱法

中华人民共和国出入境检验检疫行业标准SN/T1772—2006
进出口粮谷中玉米赤霉烯酮的测定免疫亲和柱-液相色谱法
Determination of zearalenone in cereals forimport and exportImmunoaffinity column andliquid chromatographic method2006-04-25发布
中华人民共和国
国家质量监督检验检疫总局
2006-11-15实施
本标准的附录A为资料性附录。
本标准由国家认证认可监督管理委员会提出并归口。本标准由中华人民共和国辽宁出人境检验检疫局负责起草。本标准主要起草人：荫凯、李军、卫锋、宋文斌、林维宣、田苗。本标准系首次发布的出人境检验检疫行业标准。SN/T1772—2006
1范围
进出口粮谷中玉米赤霉烯酮的测定免疫亲和柱-液相色谱法
SN/T1772—2006
本标准规定了进出口粮谷中玉米赤霉烯酮检验的制样和免疫亲和柱-液相色谱测定方法。本标准适用于进出口玉米、小麦中玉米赤霉烯酮的检验。2制样
2.1试样制备
将样品按四分法缩分至1kg，全部磨碎并通过20目筛，混匀，均分成两份作为试样。分别装入洁净的盛样容器内，密封，标明标记。在抽样和制样的操作过程中，应防止样品受到污染或发生待测物含量的变化。
2.2试样保存
将试样于一5℃以下避光保存。
3测定方法
3.1方法提要
试样中的玉米赤霉烯酮用乙腈-水提取后，提取液经免疫亲和柱净化。用配有荧光检测器的液相色谱仪进行测定，外标法定量。
3.2试剂与材料
除另有规定外，所用试剂均为分析纯，水为蒸馏水或相当的去离子水。3.2.1甲醇：HPLC级。
3.2.2乙睛.HPLC级。www.bzxz.net
3.2.3乙腈+水（9+1)：取90mL乙腈加10mL水。3.2.4氯化钠。
3.2.5玉米赤霉烯酮（zearalenone)标准品：纯度≥98%。3.2.6玉米赤霉烯酮标准溶液：准确称取适量的玉米赤霉烯酮标准品，用乙腈配成浓度为0.1mg/mL的标准储备液。根据需要用流动相稀释成适当浓度的标准工作液。3.3仪器与设备
3.3.1液相色谱仪配有荧光检测器。3.3.2粉碎机。
3.3.3高速均质器。
3.3.4氮吹仪。
3.3.5空气压力泵。
3.3.6玻璃纤维滤纸。
3.3.7玻璃注射器：20mL
3.3.8免疫亲和柱：玉米赤霉烯酮免疫亲和柱。3.3.9微量注射器：100μL。
3.4测定步骤
3.4.1提取
称取试样约40g（精确到0.1g）于250mL具塞锥形瓶中，加入4g氯化钠及100mL乙腈+水1
SN/T-1772—2006
（9+1），以均质器高速搅拌提取2min。定量滤纸过滤，移取10.0mL滤液并加人40.0mL水稀释混匀，以玻璃纤维滤纸过滤1次～2次，至滤液澄清，随即进行免疫亲和柱净化操作。3.4.2净化
将免疫亲和柱连接于20mL玻璃注射器下。准确移取10.0mL（相当于0.8g样品\）上述提取滤液注人玻璃注射器中，将空气压力泵与玻璃注射器连接，调节压力使溶液以约每秒1滴～2滴流速缓慢通过免疫亲和柱，直至.2mL～3mL空气通过柱体。以5mL水淋洗柱子1次，弃去全部流出液，并使2mL~3mL空气通过柱体准确加人1.5mLHPLC级单醇洗脱流速为1mL/min~2mL/min，收集洗脱液于玻璃试管中，再用氮气于55℃以下吹于。用1.0mL流动相[3.|4.3.1b)溶解残渣，溶液过0.45μm滤膜后供液相色谱测定。3.4.3测定
液相色谱条件
色谱柱：Nova-PakCra柱（4um），150mmX3.9mm（内径）或相当者：流动相：乙腈+水+甲醇（46+46+8）e
流速：1.0mL/min；
检测波长：激发波长274np：发射波长440~hmd)
e）进样量：50μL。
3.4.3.2色谱测定
根据样液中玉米赤霉烯酮含量情况，选定浓度相近的标准工作溶液维工作溶液和样液中玉米
赤等烯酮响应值均应在仪器检测线性范围内。对标准工作溶液和样液等体积参插进样进行测定。在上述色谱条件下，标准品色谱图参见附录A中图A.1。3.4.4空白试验
除不加试样外，均按上述步骤进行。3.4.5结果计算和表述
用色谱数据处理机或按式(1)计算试样中玉米赤霉烯酮的含量，达算算结果需将空白值扣除。
式中：
一试样中玉米赤霖烯酮的含量，单位为磨克每千克（mg/kg)：样液中玉米赤烯酮的峰面积；
一标准工作溶液中玉米赤霍烯酮的峰面积；标准工作溶液中玉米赤霉烯酮的浓度，单位为微克每毫升（μg/mi）V-样液最终定容体积，单位为毫升（hL)；m
—最终样液所代表的试样量，单位为克(g)。测定低限、回收率
测定低限
本方法的测定低限为0.005mg/kg4.2回收率
4.2.1玉米中玉米赤霉烯酮的添加浓度及回收率实验数据：添加浓度在0.005mg/kg时，回收率为73.7%~89.4%；(1)
1）对于玉米赤烯酮含量较高的样品，可将提取滤液进行适当稀释，以保证玉米赤烯酮的含量不超过免疫亲和柱的吸附容量。
添加浓度在0.050mg/kg时，回收率为76.5%~90.3%；一添加浓度在0.500mg/kg时，回收率为87.6%~98.2%。4.2.2小麦中玉米赤霉烯酮的添加浓度及回收率实验数据：一添加浓度在0.005mg/kg时，回收率为76.8%90.7%；一添加浓度在0.050mg/kg时，回收率为78.3%~93.6%；一添加浓度在0.500mg/kg时，回收率为86.4%~93.2%。SN/T1772—2006
SN/T1772—2006
附录A
（资料性附录）
玉米赤霉烯酮标准品的液相色谱图8
玉米赤霉烯酮标准品的液相色谱图图A.1
Foreword
AnnexAofthis standard is an informativeone.SN/T1772—2006
This standard was proposed by and is under the charge of the Certification and Accreditation Admin-istrationof thePeople'sRepublicofChina.This standard was drafted by Liaoning Entry-Exit Inspection and Quarantine Bureau of the People'sRepublic of China.
Themaindrafters of this standard are Suikai,LiJun,WeifengSongwenbin,Linweixuan,Tianmiao.This standard is an inspection and quarantine professional standard promulgated for the first time.Note:This English version,atranslation from the Chinese text,is solelyforguidanceSN/T1772—2006
Determinationofzearalenoneincerealsforimport and export--lmmunoaffinity column andliquid chromatographic method1
Thisstandardspecifiesthemethodof samplepreparatigumnandliquidchromatographyof zearalenoneinThis standard is applicable for the deteport.
Samplepreparatio
2.1 preparation of test
mationby Immunoaffinitycol-
tandexport.
lenoneincornandwheatforimportandex-Reduce the sampleto ca 1kg byquartering:grindwitha grinder to letall pass through a20 meshsieve, mix thoroughly and divide into two equal portions, place in clean containers, seal and label.Duringsam-plingandpreparationofsample，precayanyfactorsthatmaycausetheon
2.2Storage of sample
Thetestsampleshallb
3Method of detern
3.1Principle
The zearalenone in the test
of residue
below-5℃
d be taken to avoid contamination orDtawayfron
edwithac
immunoaffinity column for cleaninationis
Water. The extract is applied tomeans of liguid chromatographyequippedwithfluorescencedetector，usingexternal standardmethod3.2 Reagents and materials
Unless otherwise specified,all reagents should beof analytical grade,\water\isdistilled water orcorresponding de-ionized water.0
3.2.1MethanolHPLC grade.
3.2.2 Acetonitrile:HPLC grade.3.2.3Acetonitrile-water(9+1)：(90+10,v/v).3.2.4NaCl.
3.2.5ZearalenonestandardPurity98SN/T1772—2006
3.2.6 Zearalenone standard stored solutionAccuratelyweigh an appropriate amount ofzearalenone standard, dissolve in acetonitrile to prepare a standard stock solution of o.1 mg/mLDi-lute the standard stock solution with mobile phase to the required concentration as the standardworking solution.
3.3Apparatus and equipme
3.3.1High performance
High-speedblender
N-evaporator.
Glassfiberfilterpa
Glass-syringe:20
chromatographequippedwithfluore3.3.8Immunoaffinitycolumn.Zearalenoneimmunoaffinitycolumn3.3.9Micro-syringe:100
3.4Procedure
Extraction
ce detector.
Weigh40g(accurateto0.1g)ofthetestsampleintoa250mLglassmixedcup,add4gNaCland100mLacetonitrile-water(90+10,V/V)byblendingathighspeedfor2min.Themixtureextractwas filtered through filter paper. 10.0 mL of the filtrate were collect and mixed with 40.0 mL dis-SN/T1772—2006
tilled water.Thediluted extractwasfiltered throughaglass microfibre filterfor 1-2times till clearand thefiltrate pass throughthe immunoaffinity column.3.4.2Clean up
10 mL of diluted extract(equivalentto 0.8g sample)were passedthroughthezearalenone immu-noaffinitycolumn at alow-rateofabout 1~2drop/second,till 2mL~3mL airpassed through thecolumn,followed by5.omL distilledwater at one to two drops per second. Exudateswas discardedand2mL~3mLairwaspassed throughthecolumn.Zearalenonewasthenelutedwith1.5mLmeth-anoland collected in a clean glass tube. The eluted extract was evaporated under a stream of Nz at55c in heating block and the dried residue dissolved with 1 mL mobile phase. The solution filteredthroughao.45μm filterand thefiltratepreparedfordetermination.3.4.3Determination
3.4.3.1HPLCoperatingconditionChromatographiccolumn：Nova-PakCig（4μm)，150mmx3.9mm(i.d.)orequivalent；a)
Mobilephase：acetonitrile-water-methanol(46+46+8);Flowrate:1.0mL/min;
Detectionwavelength:excitationwavelength:274nm;emissionwavelength：440nm;e)
Injectionvolume50μL.
-3.4.3.2HPLC determination
According to the approximate concentrate of zearalenone in the sample solution,select the standardworking solution with similar peak area to that of sample solution.The responses of zearalenone inthe standard working solution and sample solution should be within the linear range of the instrumen-tal detection. The standard working solution should be randomly injected in-between the injectionsof the sample solution of equal volume.Under the above chromatographic condition.For HPLC chro-matogramofstandard,seefigureA.1inannexA.3.4.4Blacktest
The operation of the blank test is the same as that described in the method of determination but withomissionof sample addition.
3.4.5CalculationandexpressionofresultCalculation the content of zearalenone in the test sample by HPLC data processor or according to theformula(1),theblank value shouldbesubstracted fromtheresuitofcalculation:Axcxv
（1）
1)Etract wasdilutedappropriatelyforuppercontentof Zearalenone in sample,ensuring the content of Zearalenonewasunderthe maximum adsorption of Zearalenone immunoaffinitycolumn.where
theresiduecontentofzearalenoneinthetestsample，mg/kg；A-thepeakareaofzearalenoneinthesamplesolution;Asthepeak area of zearalenoneinthestandardworkingsolution;SN/T1772—2006
theconcentrationofzearalenoneinthestandardworkingsolution，μg/mL;V
-thefinal volumeofthesamplesolution,mL;-thecorrespondingmass of thetest sample inthefinal sample solution,g.4Limitof determination and recovery4.1Limit of determination
The limit of determination of this method is 0.005 mg/kg.4.2Recovery
4.2.1According to experimental data,the fortifying concentrations of zearalenone in corn and itscorrespondingrecoveriesare：0.005mg/kg,therecoveryis73.7%~89.4%;-0.050mg/kg,therecoveryis76.5%~90.3%;-0.500 mg/kg,the recovery is 87.6%~98.2%4.2.2According to experimental data,the fortifying concentration of zearalenone in wheatand itscorresponding recoveriesare：-0.005mg/kg,therecoveryis76.8%~90.7%;-0.050mg/kg，therecoveryis78.3%~93.6%;-0.500mg/kg,therecoveryis86.4%~93.2%SN/T1772—2006
Annex A
(informative)
Liquid chromatogram of the standard zearalenone1.5
Figure A.1 Liquid chromatogram of zearalenone
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