【商检行业标准(SN)】 出口粮谷中黄曲霉毒素BB、GG检验方法液相色谱法

中华人民共和国进出门商品检验行业标准SN 0277—93
出□粮谷中黄曲霉毒素B1、B2、G1、G2检验方法液相色谱法
Method for thc determination of aflatoxinB,,B,,G,G, in cereals for export-Liguid chromatography
1993-12-28发布
中华人民共和国国家进出口商品检验局1994-05-01实施
中华人民共和国进出口商品检验行业标准出口粮谷中黄曲舞毒泰B1.Bz,G1、Gz检验方法液相色谱法
Method fur the determlnatiob of aflatoxinH;B,.G-ti,in certals[or expartLiquid chrumatograpty
：主题内容与遗用范围
SN 027793
本标准规定比限·中黄曲解雄索H，心心检验的拍样，制利波拒色谱制定方运本标注学用下生已上中黄曲雄单本B总：心1G：的拉险。人米、小在等控谷中劳归露穿充的检资，可营聚采月本凉注
2抽样和制样
2.1岭验
以不超过23--趋销。
同一拉监批的商品宽具有构问的特征，的包装、标记、严地、规希、等继会2.2势样效范
2.2.1整贫商销核代(1)计算拍您数，e-=VN
式中：2-
一制控装整
N-全批袋数：
让：收取装教，小牙节避位等效2.2.2脱牌年，使准高度不超注2m，这稳照间区说点，以5m为个联单长，舞区设心及日第（距边线11处不点。海增买，个收大指设3个点2.31
2-3-1单计取样载：不帮调算.金长55cm位站于柄，直径1.5～2.5，沟长度盘超过装对单线使的一乎，见图1
2.3-2双套医取样器：可根不绣别誉构成，全长150m（包括手病），外管分成态个空将，内管上有继内管直径2. 02.:m,见图2,
2.3.3 取择:见图
2.3.4样：胜1，
2.3.5性品商袋资
2.3.6外性布或适销热物，
中华人民共和国国家进出口商品检验局1993-12-23批准199-:01实施
2.4拖祥方法
2.4.1较装抽
5N027793
图1单管取样器
双取样器
1316em
图3联样铲
图4分样板
2.4.1-1倒色拍样；以推垛的各部偿断机抽取2.3.1提定的应拍栏件数的11为（每挑一整不少于3包，将装口键线全部拆开，平肾于分并布或其他洁净的辅垫物1，效手紧短装底两角，强起约成45候独，倒拖1m以上，使貌内货物全部倒出。检盘货物的外观、气味，有龙发每、变适等，并查看袋内和带间SN 0277-93
品质是否均勾。确认情况正带后，用取样铲随机套各评位轴取样品，立即特样品倒入盛样器内，转发拍取的样品整应基本一致。
2.4.1.2袋内拍样：按2.2.1规定的应抽样件数的90%，在操地回周上、中.下各层以前线形走向.随机抽取，将取样器（2.3.1)件格期下，从每袋一角依斜对角方向插人橙内，始后将管槽差转上，抽出取样器，立即将样品到人盛排容器内每袋抽取的样品量应与2.4.1.1基本一致、每批样品总整应不少下4kg，
2.4.2依职拍
按2.2.2即定的取样点，逐点攻样.取样时将取样器（2.3.2)垂直(略带斜)描入取样点的粒堆内，施转外套管拍出取样管，立那格取出的样品创入妮样器内。从各点中抽收的样品量应基本一致，每批祥品总摄应不少于4kg.
2.4.3大样缩分
袋装托品荣中袋内和倒包抽样所取全部样品，做于分样布上，用分样板按匹分法缩分出样品个生于2kg，立干盛样器内，加封后标明怀记并及时送交实验室。散积样品将拖取的全前样品，例于分样布上，以下按上述装装样品法逆行。2.5试样制备
将杆品按四分法缩分垒1kg，全部磨碎并通过20日筛，混句均分成两份，装入洁净的奔器内，案封-明标记。
2.6试择保存
将成样于5C:以下证光保存，
注，在栏考积出当的减作过留中，必须快止样品受割化单或发生贯重需海来合整的化。3测定方法
3.1方搭提要
试择中黄曲要毒素用二领早烷提取，提吸液氧化铝和建胶桂净化以除去脂肪色衰及杂质，月液相色谱法荧光检测器测定，外标法定尿3.2试剂和材料
所用试剂除注明外均为分折纯，水对蒸谣水。3. 2. 1 二氯甲烷。
3.2.2莱。
3.2.32嘴，
3-2.4正已。
3.2. 5无水乙醛，
3.2.6丙酮.
3.2.7乙胶。
3.2.8无水甲酸，
3.2.9死水甲炉：
3.2. 10乙酸乙酯,
3.2- 11 甲苯。
3:2.12水硫酸销：统650心灼热4h：胃于干类器内备用，3.2.13巾半氧化铝，层折用，粒准100~20V日经30℃活化211.还于下爆器内备用。3.2.14硅胶.晨款用，粒度60~325日，疑110℃括化2h并加12%据合3
sN C277 93
溶剂定容于[00ml棕举或塞，配或标准能条控游，浓流可：7m/mt.移上连四种添准储备淳液技BBGG4..体比充逆于1穿中度依次B0限mL.B3.0/mlG，12.0+g/mLG，：1.0ug/ml.，起落波为瓶合标能储各落效，根据游要再配成适用法度的标准工作溶液。
33仅需和没奋
3.3.1液相色谱仪，配有炭光检测器。3.3.2粉碎机。
3.3.3长端%
3.3.4提转蒸发器并配有150mL..250mL底烧瓶，3.3.5现器。
3.3.6滤膜：有机系用，0.45m。3.3.7微孔膜让滤器，有枕系用，n.6m3.3.3层杜：8rmm×20mm（d）玻离主，下具适塞，在前底密人少减破转乌将5g元水统酸纳，1R硅胶、5g中性化铝，15多无水镜酸钠的原序收次出亚已势湿福器柱，3.3.9进择瓶,2mL波率瓶，具密对笨，3.4测定作
称取粉摔温勾的试样30g（精确到0.1g）乎360l.具键形，确入二点甲满130mL在标荡器上搬提取30mir以快源定性滤纸齿除清液50mL于150ml.两晓中（过微时灌斗二加益表获离，以防止格剂撑发).用旋转凝发器在50亡水浴中将诺剂蒸发至竿，3.4.2布化
上述残整以5mL二氟甲烷-正己烷（1+1)混合游剂容解产移入层开倍，四腐烧施再以5mlL同拦溶剂待添一次，活保液并入是析性。打升忙下活塞，技之自然末下，待液面接正是而粒上层无水硫酸纳表而时，低次以80mL苹-骏（9+1（流独2~mL/mia15VL元水乙-已（-1）流选小~5ml./mir)分游洗层相在，弃去流出波。最后以20m>mT，：甲烷-丙酮（3—1)流随5-6ml/am)洗脱层折柱，以T.底您瓶承接流脱赖用旋转东发器在55℃水举中将上述洗脱然发至，用1.nL甲车水甲好-乙酸酷（90+一能合孕剂案解残产，并偿其通逆造孔滤端滤器收集于进撑辉宁供波相色诺测定。3.4.3
3.4.3.1色谱茶件
，色带坐：，5-250mm×x4.6mm（d）：b.动东-元水甲醇乙酸乙嘴-水甲碳88-3+十2）。肥制所经J.5m湾滤，税气（他时当天配制；
.筑速-..1.min
d，激发肢险 3G5 rm、激发狭10 nm;发计波理-25 mm.发射疾链 1m t-13.4.3-2运谱测定
根培样液市黄灯零海索含适性识，定峰高相近的标障工作游液，格作工步滚策详液中成拍游部素询应宜均应在独检测线性范围内。对标准工作溶液和偿波等车积出还造行游盘，在述色照杀件下,黄曲群牵素的出唑显序较保照时间次约为 B;> rin,R::1= :min,G,:3 rin,C:16 min,3.4.4案白试验
综不加试样外，坡上述源定求骤进行。3.5结果再新表述
用积分仪按外标法相关伤库分测计产黄曲举牵案B民，、，垫，成按式3房行计算，式中：X
SN 027793
分别为试样mt黄曲飛标家BB.Gt，的含缺，gkg为一分别考样液中曲军毒诱B.B：G.G,的药m分别为标准工作窄液中黄出将索案D，B，G，C，的锋高，mm分别为标能T作落流中黄曲毒牵素BBGG的浓度R/1V-择液最经定群体也！ml
爱终样液所相当的学品成
法：计放结果需北除空日面，
4测定低限，回收率
4.1测准低限
本方法的就定低限为：
Bt:h 50 μg/kgt
D.:0. 3R μe/kg:
G.:0. 25 μg/kg:
G,:0.12 pR/kg.
4-2向收率
回收率实验数据
浅游度ig/kg
附加说明：
0. 5C-~5. 0
0. 38-3-8
0. 25.~2- 5
0. 12 1. 3
回收率，%
76. 7-92.2
68, 5--99 5
68. 1 --100, S
6.9~-101.6
本际洋的出作人以共和国国家进出商品检验与提出。本标陷中华人民共和国天作选出口液品检独局负资就账。本标温主要起人郑生，张洪卫、质期。(2)
Professional Standard of the People's Republic of Chinafor Jmport and Export Commodity InspectionMethod For the determination of aflatoxinB,B,,Gi,G in cereals for export-Liguid chromatography
1Senpe and field or appllcatlonSN 0277-93
This etatidaed specifies the nathod al samplurg,ganple preparation sr.d determination oy liquidchromatography of afiatoxin Ey,H,,G,,C, in cereala for cxport.This gtaadaid is applieabie o che deicrmination uj fliutoxinB,B,G..G, in uaiaa Inr expori,Thiestandard caa be adoptrd for the detrrminutiru n aflasoxins in :ice ,wheat ard other cereals for tete?Rnec
2 Sarapling and sample prepatalion21Inspcerion lat
Enech inspeciin ll shuuld uat exceed 20c metris tonsThe characteristica of the cargo within the same inspecton lot,such Ea pack.ng: ark rurigitspecification and grade ercghou'd be the game.2.2 Quar.tity of sample takcn2.2. 1Curgo in bugs
Caleuiate. -he nunber of bags to be tasen by tormulatl)rwl.ety
d :: nu.ber of hags to be teken?N.tatel oumher of baga iny lotQ=VN
Nuse: value u is with dorimal,round sff che dec:dal pert, which fs added sa enity \g (he intrgral pan nf a.2.2.2Carzo in bulk
Tlhir leighi of rhs rereal pile should aot exceed 2 m. Set up arers and spors for sampliag on thepile .riace.S m is ennsidered as an area .in whicb 5 spor shall be fixad anr in th rentrr Bad for.a: Eour carrere,but 1 m fram the margin of the μree.For an additional ara,3 mre sampling spdshould be fixed.
2.3Sernplingtools
2. 3.1 Mr1aliu nurplnr,Stainlese etecl tube riengthtineluding hsadie>55 cm :diarocter 1.5~-2- 5 cm:groove length longer than half the diagonal length af che bagtscc fig -1.Approved by the-Stute Adminisiration of Emport andExpart Comaodily Enspeclion uf the Peopte's Repubticuf China on Dee.28.1993
Tmpleraented frumMay.t.1994
5N 0277—93
2.3.2Double-easing Ruispler: Consists af two stainless steel tubes: length 150 em (ineludinghanitle ),dianeter of inner cazing(wich grogve]z.O~-2.5 cmiouter cusing having 6 elntssee fig.2.2.3.3Sampling shovet:see fig.3.2.3.4Plate fos quarttring :zee fig-42.3.5Samp'e eantbag?,whiuh carr hetated2.3.6Clas (or other material)sheet:For aample dividing(qusrterirug)cn
fig- 1 Metullin xampler
fig. JoualeEausing ianupler
fig. 3 Sampling *hovel
fig·4 Plate fer quartering
2:4Sampling Erorecure
SN27793
2.5.1Sainpling lor the cerals from the bagy2. d. 1. 1Sampdiag hy ujulyirg iar.)raw 1n percent of the number of bags specificd ic. 2. 2. J(nor lexx than 3 hag+ at any part of thePd: t :andur.. Uneeum, aid open ihe bug,and erecr it n R clenn elora sheet (or other clean shcet).Grasp ught two corners af sa bug bottom and ruis, u[? to ar, atgle ul 4s'.tug hark wari far La I m la-rd all rnirml tif :hie hiy is crazticd out.Check the appearance.odour of che goods and whether thegords are maldy,ratten ete.Alsu check whethe: the quulity ot the goods is uaform within and be-twcen the bags.Alrer conlirming tae gnad a:t in normal eaudirior.scsop up the sanple from diffcrcntparts of the oat pouredl epntert witt: u shuvel ,unl pln. in rt hirpee ontaint Pernuply, Th tunelitycf th xarrpl- drwa [rim each bag saoald he hasically tae same.2. d. 1-2Sanpliag fo= the cerea.ls in the bagyLraw tle ssmples frnm D5 pereent uf the numher : lwig% njirtfinit in 2. 2. 1 as fi:l siws:Alang 1lesine wavu uf ilhe rile.driaw win:pl frmm lle hak- of he .pper,nniddle and iower percs of the pile atrisuiort, Tasell the eampler(2. 3. 1),with its gruove facing cowaward,ciagonaliy :nto ach hag-thetura the xetnpler hy 1so',draw aut rhe samp'e and pamp..y your -ac sample iato unaincr. Thequantitg of the suran'e drawu fem och bng nliunl.f l. luairally le saru as it 3. 4. I.1.Tine Ilal we'ghi nf ihe reprerentative simple cf eaca lnt shculd be not less than kg-2.4.2 Sumplicg Ier pile ia bulkFix tine aamolirp sors as specified in 2. 2. 2.draw tae sagles Irom the sruts sueesively by in-acrtsng the sampier t2. 3. 2ivurtiual'y (ulightly icutinul)ir tt ltp t.rintp sf e pile, Turn tlie nu.er c.a-sirg p:ll >! ili hilupier,and imnediatuly pour the sampie into the samp'e centaincr, The quz.ntity ofthe samp.es drzwa fram all the spats sha.l br baxically the iyre and the tatal weigh: of the reprasru-Ialive sanp.e af each lor should be rot liss then I kg.2. 4.3 Reluetion f grun s inple.Far 3. d. 1. Fnur all the sanple (irom bocs 2. 1. 1.1 anc2. 4. 1. 21un a cle2n ztee1.reduce to notless than 2 kg. witt. a pate by q'arierinig. Hlace in sampie coniaiaer:heal.label and aend t the labara-ry i lire.
Fur 2. l.2: Post ell :he aplr nn p re aheel ar.. carty aut the Atahsne;cent Frocess as that Inrilir suraplr I:rin ilie bat? cescr:bed abovc2.5 Preparation ot test satpleReduce the semple ro se J kg by quartaring+grind thuriyaghly end let ur thrinugh id 2c ttuxhsicve ,mix wull,d:vide intu 2 ucual ag-tions .pl.re in lrisn rinta.nra,seal aad late.2. 6Strgf 1+ ple
The teet sarolc: shall be scuree Lelow 5 C and kepr rvny isom lightNglr: [n lh suurzt ol +ellpliny ead sampis preuration precautic. r.u:t he taken ta avoid ccntaninarinn us nryIaelits u l.icei y czuse th- cgs of ailatovin xnent3 Mrthod ol determinalion
The ailatuxins sre rxtraeled rom the aample with dchlorsmctharc ,tac rxiract is cleed ep by a-lunrea-silica gel colunua to renuv ths far.jigrun und intn-fering alslampn.au. laternainel byL.C wuth Huuruseutw rietutl,t.usi:g rxlerual tandard methn.S
3.2 Reagerts and matarial
SN 027793
Ail reagenrg are of ann.,tigal g-ale unlssx atherwise saceified warer is cistilled waler.3.2. 1 Dicklsretethene-
3. 2. 2 Prmazene.
3.2.3Acetonitrile.
3- 2. 4 n-Hexare.
3. 2. 5Dpyidritlel eibyl etlirr,3-2.6Acernne.
3.2.7 Acst acic.
3. 2. 8 Drhydratedl formic acid.3. 2. 9 Ienydhdled mess.nnl.3.2-10 Ethyl 2cetate
3.2-11 Teluenc.
3. 2. 12 Anityit:auk srilielm sullfae:Tgiuitl al ter: C Eor 4 ht.nd etcred in cesiecaror.3- 2-13 A.urriga, neutral, For chroraatigraphic mse, 1an-7nn n:psl, a:-liva re: a: 13(t c fnr 2 1.stored n ceaiccator.
3. 2. 14 Sili.: grl, F:r rri;rm:lugapeit un.6u.-325 a.csh: Hcveted et 11c C fo: 2 h,aad rhea ce-2ctivated wi-5 ::(m/ni)uare:-3.2. 15 Aflataxin H,,Eb,t,,G sandarde:Parity al eachb:y:3-2. 16Afluloxin wtundurd soluriuns,Weigh 1. J mg of each eliatoxin E,.H,,tj,,Gr anil nisxaive zepat-Fntely witk hencene-arntnuirile (9s+e) mixlur in lo nL hrown voiume:rv: flagks End dilute co vei-iume as tte stardard stock solutizns.The coacentration of each stock aolutim in id juru/inl.Mix tin.standard scock solutiona in a 10 ml, brown alumetric Ilask with the velume tatio pf 4i3-2:1: n ibe iixril alartiard slock soiusior. The runeentretions of dfezanc ailstoxias in this anixvislndaril nlock saluliul ar H,:4.ll fu/miL,2,:3.unn/reL.G.:2. pm/mL.C,:1.0 am/m, reape..vejy.According to the recuirement.ereparza mixeid stanliar!wtsk.ug nnlutiga of apgrupriaiu conunralon.
3-3Aarauskndcprt
3.3 1 1raid chromatagraph,apiprl winh fuuruseue detceor.3. 3. 2Gtincer-
3.3. 3Shaker.
3. 3. zRi.tary vapraator wil. 15o mz.25u I. rourc.bostom flask.3. 3. 5[ilass sacuum fil-rat.or. devire.3.3.6 Fltration rembane:Fo: rganis solvent .C-1F gm.3. 3. 7Microporo.is matnbrare flter ;For organic soivent,C. 5 μti3. 3. 8Clirn.ntgrapfi: t'unnt3ci nirn X2r mm (id>glass colunn with. 5togcock.Paak the colurrEn the follnw.ng ordersa eraa'l elag of glass wanl ut .in lulturi,s g uf unllyclrau swdiurs suiface, Jc. goi si.ica gel.5 g sf neatral slurainu. lc g cl anhydrous sodium su:fate .the reagence heing ruisi rieulwi.a n-hexaae ws.le filled in.3. 3. 9Srrip'r vial:2 tnL.wi.:i light cuver3.1 1'tedurc.
3.4.1 Extastior.
Weigh. so Rtaceurare co o. 1 g>cl the mixed ard grgand tes: sutnple icto a 2o mi. Eranney..5
SN 0277—93
laek with ztopper.add exactly 100 mal. of dichlorormethane and extract for 30 min on shaker .Filterthrough rapid quantitative filcer paper nd traasfer 50 toL of the clear filtrace inta Is0 muL ruundbottom flask (rover the funnc) with a watch glagg while filteriag to avoid evaporation of the solvent).Fvaporate ta dryness in a water bath ac So C winis he rolary evajruratur.3.4.2 Clean up
Disaolve the above residue and tranafer ineo the chromatographic colurun with i ml. ol the mixedsrilvent of diclloromerhne-n-hexanr (1+1),the round.bottom flask is rinsed with anothet 5 mL ofthe same mixed solvent,and combining the rinsing into the coluinn. Open the stupcock of the column,let the aolution drain freely, Whe the surface of the solutipn reaches the top nf the upper ain?tydroussutliumn suifute in the column,wash the rplumn seguently with Bo mL of beuzene-acetic acid(g+1)(flow rate 3—3mt./min)and 150 mL of dehyrlruted ethyl ether-n-haxane(3-1)(flowrate+5mL/tmir.).Discerd tae ellluentz.Finaliy,elute the calumn witl 2on mL al dichlurumechane-Bcetane(g+1)(flow =are 5—6 mL/min),and the eluare is cotlected in a 255 mL round-boccoru lask.Evuparaie the luutr 1u lrynexs in a watet bath at 50 Y with the roraty evaporator. Dissolve thereaidue wich 1, 0 mI, Df the mixed mlvenl of loluerie-tdehydrated methacot-ethyl acetace(g0+2+8),filtet through the microporous membrane filter,and collect in a aainple vial for LC unelysis.3.4.3 Detcrrinatior.
3.4.31C rperatinug tonditicra-Chromatographic columrSilicaxel,s μm,250 uitnX 4.6 mtatid):b.Mobile phagerto.uene-dehydrated methanol-ethyl acetate-dehydrated formie acil (88+2+8+2),filtered under vaeuum thraugh 0. 45 μm membrane.and degassed(prepazed freshly caily),Fluwratel.umL/min:
dExeitation wavelenwth 365 nm,excitalion zli1 10 Tii;Erissiun wavelength 425 nm,emissionslit15nm.
3.4.3.2Lc determinat:on
Aruanlinig tu the cuntents of oflatoxis in the sumple solution,gelect the apatoximate standardwarking solution wicn kimilar peak heights tn thone of sample sulutiun.Tie resyongea of afiatoxins irthe standard working slution and sample solutipn should he in the lineut ringe uf the iwtrimentaldetection. The standard working solution should bo injocted in-between occasionally with the anplexplutiun uf cqual eolurme. Under above condition the eluting seqwense and the approximate retentioniinexal aflatuxins areB,min.B,.llin,G.13minGrl6min.3.4. 4lank tes!
The operatipn af the blank tes: is tae same es thaz dercribed in ihe: trirliurl f rlelernsiruition,buewith omiasian cf sampteaddition.3.5Caleuiscton end expreasion of regul:The caleulation of contents of afiatuxin B,,B,G, G, can be aarried our, by relevant prograrmmes ofexterral slaudnrd mwthnd in thr initegratur,ur by fomulat2):X
×1000
X—the respeative enntents of aflalnxins B,,t,.G,,G, in the trst sarmr:lc-pgrkg!—the reapective peak heighc of aflatnxine ,B.G,.G,in the niumple sulutian,mm;10
SN 0277—93bzxz.net
h,-the tspetive pek heights of allautuxins B,, Bz.G+Gz in the scendsrd working solutwn.itnE
—che reegective concentrations of aflataxins B,.E,.G.,G, in the taudard working solution,2g/ml1
V-chefinai volume af thesamplesolution,mlmthe carrxpondiuy aas of tlie teet baliple in che fina xample ealucionh.Note.: Fhe hlank valur shnuild he nubzrarted Eenr. :he zolenlntrd esulr.4Limit nf determination and recavery4. 1 Linit af determination
The limits of daterminatio ot thie method are B,-O.SG μg/kgtB,—0.38 μg/kRt
G.—0.25μg/kgF
G2—0. 12 μg/kg-
4.2Recovery
Expcrinventail recovery dala:Alatoxns
Faetifyiag
coRcencEB-
ion+g/kg
0.50—-5.0
0. 252. 5
Adallisml explapntione :
Recnvery.%
68.6—93.3
66. 7—100. 9
65.0—101.6
This stanlarl wus grppased by the Stuie Administration of [rngurt atial Exjurt Cummadicy la-sjection nf the Peopln's Republic of Clhina.This standard was drafted by the Tianjin Inport and Export Commodity Iaspectian Bureau of thePeople*s Republc of China.
Tlhis standaril witn aialy drafred by Zheng Honugsheng.Zhung Hangwei,Tang YiNote :'This Fingl.ah vrraionae transletlcn from the Chincse texs,ia qolely For uidaner.12
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