【商检行业标准(SN)】 出口茶叶中黄曲霉毒素B1检验方法

中华人民共和国进出口商品检验行业标准SN0339-95
出口茶叶中黄曲霉毒素B1检验方法Method for the determination of aflatoxin B.in teaforexport
1995-05-29发布
1995-11-01施
中华人民共和国国家进出口商品检验局发布中华人民共和国进出口商品检验行业标准出口茶叶中黄曲毒素，捡验方法Method for the determlzailon ot aflatoxla B,intetorexport
1主题内赛与源用范围
SN0339—95
木标准规定了出口苯叶中黄曲再毒素乃，含量检酸的抽打，制样和被相色谐测定方法，本标准适用下出口茶叶中黄曲等素，含基的捡验。2抽样和制样
2.1检验批
以不超过000件为：检验批。
同一检验挑的商品应兵有同的特征如包装，标记、产地、规静、等级举：2.2捆样数量
批基，件
51~-500
1 001-~1 300
1 501--2 000
2.3拍样方达
敏抽样数，件
以性批产品堆珠的上下不同部位随机拍取2.规定的件教，逐件开启：分别出全部茶叶十塑料布上.用取样从萍件中告取山有代表的样品约5。将所取样品充分泥分，用四分法或分样器逐步缩分出5装入沾密均的竹吊商内加封后，标明标记没时送实验案，2.4试样制备
将所取回样品金部率辞，过2C日临，涯匀，期分成两份试样，装入法净容器内·密判，标明标记，2.5试拌保存
将试样了室温下保行。
生，抽利制的性过理守，必质止主品受更乘或效生设密物含谢的空化。3测定方法
3.1方法抵要
详品用三效甲烷添取，损取薇经律胶样净化，你化后的折版商用子行生，用配有获光抢别光的减祖色前仪测定，外标法定所3.2试剂和杖料
中华人民共和国国豪进出口商品检验同1995-05-29批准1995-1101实施
S0339-95
所月试润，除持殊注明外，均为分析纯，水为重蒸馏本。3.2.1三载甲烷。
3.2.2正已篇。
3.2.3 萍。
中醇紫外光谱级.
乙腊：紫外光谱綫。
3.2.6三氯乙酸.
乙情-水溶读(111)
3.2.8二中烷-中醉裤箍（95+5)
3.2.9半乙腈溶（98-21。
3.2.10黄出等寿B，标准品，纯度9%3.2-11黄曲带毒B，标准液准确称收道低的黄曲需牵素B，标准品，以举-乙清游滤（3.2.9)丁棕任穿其忆中，配成浓度为10m上标准吧备液，根据需要，再配成适当法度的标准工作游液。3-3仪器利设备
3.3.1液柑色谱位,配有荧光检测盟，3-3-2硅胶小杜；watcr=Scp-pakSiica前处理小社或相当的硅胶雅处理小柱，3.3.3振端器
旋转蒸发器，配有10)L其路管的医庭烧瓶3. 3.5
3. 3. 6 离心管:5 ml. 宽善。
3.3.7粉碎机。
3- 3- B 滤膜,有机系用,0. 45 tm.3.3.9微孔滤膜过泄器：有机用，Ihn。3.4测定象
你取试样5.0（势到0.1g）肾于100m具座铺形烧瓶中，加入15m件烧，于报荡器上提来Umin，然后经垫有被纤维的游斗过滤。改典滤减于旋转蒸发器的其民单回麻烧瓶内，井用三书甲烷洗漆滤连，收集滤液率约2m1.3-4-2北
用施转黑发器游上述滤箍在5C℃水浴中依缩车约1mL，经.45rm滤膜造滤后，注入硅践小中。月2~4m正二始洗涤烧瓶后淋洗小托，奔虫洗出减，然后中3~4ml.二中烷-甲等洱商（3.2.9）以每秒钟2满的流速洗脱，收集洗脱液了离心管中。用微气缓收十，班衍生用，3.4.3衍牛
3.4.3.1试样
加205正已烧和50三点乙酸于上述心件，灯率口准，超振诺1min，静肾13mim打开磨口肇，绥级证入疯气至下，用乙-水落获（1-1)（3.2.7)定容至1.(ml超声1min附U.5Jm滤膜过滤，滤浓其减相色谱用。
3.4.3.2标准工作游班
敢1.0mL标准工作游减，月过气耀短次干，按3.4.3.1步探操作.3.4.4测定
3.4.4.色者条件
a.色谱柱：OVAPAKC302mm×3.9mm(内径)。b：流动相,中醉-水锌液(42+53).2
流块0.8mL/mn
SN0339-95
荧光检测器，激发波长375nm，发乐液长425amd.
自谱耗辐度：室摄。
3.4.4.2删定www.bzxz.net
根据挣液守货典爱费案B：的合含盘情记，选定峰商摄近的标准工性落滤，标准工作落滤和烊液中费曲琴毒帝B，生物的响皮借的应在仅得检测线性范用内，对标准工作落减和绒的待生物萨液等体积参插进样测定。作上这色错茶件下，费曲毒事素B，衔生物保图附间约为8min.3.4.4.3控户试验
除不加试样外，按了深洲定步骚进行3.5站果1算和表述
更色谐数据逆理机成度下式让算试样中费曲器变素B，的会品，Y
式中：X—试样黄山存*家B的字量，m,·kgA一样波中黄出每率索B，行牛物的非面积的扣\；—标冶上作溶液中黄曲毒素B,的浓康R/ml.A.一标准工作溶液中营曲率素B,生物的峰面积,mm：股终样液所代表试样的浓度：/L注：计其结果需打除空白。
4测定低限、回收率
4.1测定限
本力运的测定低限为n.oln/kg。4.2收率
可收本的实验数据，费邮录系B；的涨加依度在C,01~0.5mg/km范用内，回收率为89.1%~.104.9%。
附加说明
本际准日中华人民共和国国家进出日商品检验局提出：本标增中华人长共和国江西进口口商品检验民放责起节。本标准主要起草人徐文害，黄小明、随小那。Profcssional Staadard of thePeople's Kepublic of Chinafor Import and Export Commodity InspectionMethod for the determinatipn nf aflaiaxin Bin tea fur exparl
1Scapc and field of appllcatienSN0339-95
Thie standard specifies the methad of sampliugaample preparation and deterntinacion by nighperlnruence linu:d chromatograplhy of aflatoxin B, in tez for export.Tnis star.darc is apelicable to the dletetmination of aflatoxin B, in tea for cxpurt.2Soinplingaud sampfepreparatinn2-1 . Inspuution lur
Faurh :napertinrt lnt should uni exceed 2 Gao peckages.The caaracteritics cf the ceigo wirhin tbe same inspertian lot,such as peckingrmark origin.specification art grade,should be he game.2-2 Quanticy f sampde trken
Qumalutidy in an lot.
parkaxer.
6 --50
51—500
501—1 0G
10011500
15012000
2.3Sample prpeedurc
Minimum quantiiy in he takenpackaRee
Druw the numbur uf puekagus speuifirrd it 2. 2 al tlilrrent wrts rf ilue pile eit Tuulorn. Open ertlcmpty uitl all cunieuls sf harh patkage nn u elan plastic slemt.Toraw he Tprhuenlalive su:snplr frnghe reapecrive packages,the quantity of the earple drawn Irom each package shouid Dc about fioo g.Mix and :educe tne mixed representai:ve sample lo ca 5o0 g by guarterarg or by a riffle aanpler piacein a clean conzainer which s then sealedlabeled and gcnt to laboratpry in time.2,4Preurution of test smnpleGrind all llit: salnple zharoughly and lez pare through a 20-mesh sieva,divide intn 2 eiyual nrirtiana.Place iu clean cantaineri.seal and label.2. 5Siorage of tes1 sample
The trst semple should be atared ut Iuorn trnueraturNore: Jn ili zowrse of ezr.plirg and sampl preparation.prerkurion zuust be tekon to sroid zoniarinetiou or BnyFacturrs wtirl luay cuae Jie cbaDge of regilue oiltAppraved hy the Ntate Adminkatratinn nfImprl and Expor Cammodity Inspectlou afthePeople's Republle of China on May.29.1995Implemented tram Nnv.1.1995
3Method ot deleranmation
3. 1Pineiple
SN 0339—95
The aflatoxin f, i extracteil frnm the sainple with trichlriromethane,tlie nxrint:l ix rleancd up byzilica ge! colurnn,thon the cleancd-up extract is derivatized by trifluoroacecic acid aad delerminaLel lryHPLC zith fluorescence detector.using exrernal atandard method.3.2Reagerts End ateriuls
TTnlesf ntlberwise specifil,all reageats ate analytical grarle, water ie redistilled wktet.3. 2.1 Trichlororethane.
3- 2-2 n Hexane.
3. 2.3Kellne tle.
3. 2.4 Meshann!,tJV rrade.
Acetonitrile.Uy grade,
Trifuoroacetie acrd
3.2.7Axdonitrile weler solution((1+1)3.2.8Trichlnrrimetlane-nut:hamsi colucion(es+5).3-2.9 Benzenr acetonitrile solution(98+2>.3.2.10Aflavoxin H, standard:Purny-99X.3.2. 11 Aflutuxin B, standurd aolution: Weigh a auitable amount of sflatoxin B.,and dizaolve wicabenzene-aretouiirile kolutinnf3. 2. g)in hrown vulummelrir: f'usk bnd dilute to form gtandard gicek go.[ution of 10 μg/rml., Accordinr to the requirennent,prepare 1he standard wurking ululio of sppropri.aieconceniration,
3. 3A[arutus and equigment
3. 3. High Eerlor tulmre liruicl rhroallogsaph ,tyuinpedi wita fluorca -ccnce detectpr3. 3- 2 Silice gel cnlumu : Wztere Sep-pak Silit:a cnlunin ar wyuivalenl.3. 3.3 haker.
3-3.4Rorery cvaporator+with rourd.Eettom flask with 1o0 mL ot prctrudet ti2.3.3:5Micro-iujec:m.
3.3.6Centrifugetube;5mL,withgrpundstopper.3.3.7 Gtinder.
3-3.8Filirtion membmnefororganicaolvent),0.45μm.3.3.9Micropotousmenhtara(furarganie.*ulvunt),l.pm3.4 I'rocede:re
3. 4.1Excraenn
Weigl: S.o g (aceurale tw o.1 )ol the teat saraple inta a 100 roL E:lenmeyer flask with etapper,edd I5 nsf. 1ricillsronmeluane aul rxfrirr frar J min on the shaker.Filter through a funnel with somefibrcglags and collect te clear filcrate in the rrmndl-lvsitom ilausk (with μrotruded tip)of the rocery e-vaporator. Wash the residue with trichlororuethane,tho total volume of the filirale in albout 2o rul..3.4.2Cleanu)
Evaantszt. ilen abive fili rute tw wbout I mL ia d watar beth as Fo'C wish the rotary evaporator,fil-ter thraugh a 0, 15 gμr membrane and tranefer inir Sep-pak Silica culunn,rinse the flask then the col-umr with 2-4 mL of p-hexane znd ciscard the efflucnt.Finally,elute the coluun wich 3—4 mLtrichlorpmethane-mcthenol(3,2.8)(flow rate 2 drop each gecbnd),and the eluace ia collected in a (at)SN0339-95
trifuge wube.evaporate gently to dryncaa with nirrogcn currcnt.3.4.3Dcrivatization
3.4.3.1 Fur kanuple
Add z00 μL of n-hexane and 50 μE of rritluoroacetic acid into ihe above centriluge tube, Covertightly and vibrste by uleraspnic tneang for I min. Let stand for 10 min+apen the cover of the tube.andblow gently ta dryness with nitrogcn rurreut. Divsolve th: residue with l.D mL of acetunitrile watrrsulution(I+),vilirale in by tltrasnnic means Iar 1 min,and filter through the 0. 5 μm memheane. Jil[et,ant colleet thefilsrate in a zample vial for Hl'LC analysis.3.4.3.2For standard working aolutionTake 1. mL of stEndurd wurkig Nolulinm,evarile lo lryriexx uruler irugert tut ,ttud jreed es i 3. 4. 3. 1
3.4.4 Teermination
3.4.4.1HIPLC operating vonditionChromatographiecoluran,NoVAPAKC,3commXg.mm(id)a.
b.Mabile phase;methanol water(42-+58):Flow rale:n.& ml./nsing
EluorescenceDetector:
Excitationwavelengih:375nm,
Emis91onwavclcngth,425tim:
e.Culumt tepurrulure,Roun ternperature.3.4.4.2mtrrmina1ian
According to the pontent of aflatoxin B, in the sample solution,select the approximate statudardwarking solution with similar peak height to thar of garuple solution. The responses ol aliatoxin yderivarive in the atandard golutipn aad samyle salutiun should be in the linear ringc of the instrumenTal detection, The derivaiv: uf xlaridard wnrhing nolutinu should he inj:leri ranuroaly iu-belwren llheinjertinns nf ihe derivstive nf eanple solution pf egual volume. Lnder above condition,the retenciontire of allatozcin B, derivaive is abou & min.3.4. 4. 3Elank test
The Dpcirtiul uf thu blank lest is the Aanie as that desceihwd in tte metinend sf delerinieralinsn ,lulwith unisxikui nf sample addition.3. 5 Calrulation end expressic al resultFhe calculation af cantent af allainxin li, csn be carricd put by a data prpoThe following formula.
X-Contentofallatoxin E,intestsamplem/kgiA.. Peax area al aflarpxin B, derivative in aample solucion,mm*;c.Concentretion uf aflatoxin B, in stundrd wurkirg soluuion,g/ml:A,.-Peak area nf aflatoxin B: dcrivative in standard wrkinn solutian+mm*,sororcaleclatcdbs
Corresponding concentratinn af thetest gample in irg final golutian+/mLole: The. blpnk vzlur xhould be suburacted frem the calculated result.4Llmlt of determllatioa aad recawery4. 1 Limin. of determinationSN0339
The limit of delermination of this method is O.ol ng/kg4.2Kecovery
According to the cxperimcntal dara,when the fortifying concenttation of allatozin B, is in therangt or n. on]—0.5 ing/hg.ihe neunvery is 89. 1%-104.9%.Addlonal exptamatlons:
Tlhie 6tandard was prnpneed by the State Admirnisiratien nf Iruport and Export Commoditp In-spection of the People's Repubilic of China.Fhis 5tandard was drafted by Jiangxi iniport and Ezport Commodity lnspectian Bureall nf lePcople's Republie f China.
Tlin stalulard war mninly dyadierl lry Xu Wellyin,Huank Xinuming,Shi XinoshanMote, 'This Htglinial yutninn,n Tasulstitin frnm the Chir rxe tret,is nelnly lor guidenle.
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