【商检行业标准(SN)】 出口肉及肉制品中雌二醇残留量检验方法放射免疫法

中华人民共和国进出口商品检验行业标准SV 06641997
出口肉及肉制品中雌二醇残留量检验方法放射免疫法
Method for the determination of estradiolresidues in meats and meat products far expnrt.Radioimmunoassay
1997-08-15发布
1998-01-01实施
中华人民共和国国家进出口商品检验厨发布5N0664-1997
本标准湿按照GB/T1.1一1993标准化工作导：第1单元，标准的草与表述规则第1部分，标准端写的基木规定及SNT00011595出口商品中衣药,兽药残留盘生物毒素检验与法标准编写的基本规定的要求而进行瑞写的，其中测定方法是在原科研数采的考上，参照国内外有关资料，经改进并验证后制定的。本标准同时龄定了描样和制样方法，测定低限是根据国际上对内品中雌二醇现留基的最商限量和到定力法灵敏度而判定的，本标准由中华人民共和国国家进出口商品检验高提出并归口，本标准起单单位，中华人民共和国天津进出门商品检验局。本标准主景起草人刘克,张尽大，李创影，成玉风，前润民。本标准系首次发布行业标摊。
1范围
中华人民共和国进出口商品检验行业标准出口肉及肉制品中雌二醇残留
检验方法放射免疫法
Method for the determinatina at estradiol realduesn meatsand meatpraducts forexpprtRadiaimmunna5say
SX0664-1997
本标准规定广出口肉及内制品中雌二醇残图基检验的独样、制样和放射免疫测定方法。本标准造用十脂口冻牛肉中燃二醇残留孟的检验。2抽样和制样
2.1检壁批
以不超过2500件为一格将批。
同一控腔批的商品应具有指同的特征，如包装、标记、产监，规格和等级等。2-2拥样数垫
批查，件
26--100
1G1--25C
251~5tn
501~1000
1001--2500
2.3抽样克法
最低抽样数，件
按2.2规烂的抽样件数随凯抽取，逐件升真。从每件中至少取一费作为原始样品，如每性巾无小包装成有小包装，但袋小包装平超过2，划可用灭菌刀具从每件中割不少于1混台后罩于请容解内作为滤台原始样品，很贪原始样的总量不心丁2k，加封后，标明标记及时交实验室，2.4试弹制备
从混合原始样品中取出部分有代表性样品，将可食部分效入胶库机中绞辞充分混勾，用四分法愉分至不本于5C06，均分或两份，装入满活容馨内作为试样，圳封井标明标记，2.5试栏保存
将试样于一18么以下保存。
，在抽样和部详的慢作让幅中，必额防止样品变到污势或状生政物含量的变化，3测定方法
3.1法提要
中华人民共和国国变进的口商品检验易1997-08-15批准1998-1-01实#
S06641997
试偿中残科的唯二醇，用二减中烷提收。提收液经蒸十，残道用甲醉术落商没出，没出游经性存化后，加入雄二解抗伴皮际记的增二醇格波，尔4心净活加分高，高心·最上清液，用液外闪炼分析疫避行测超，
3.2试剂和材料
除另有规定外.试剂均火分析纯，术为再蒸烯水，3.2.1磷酸氢二钠诺减：取35.81B磷酸玺二钠(VaHPO，，12H.0）路解于水中产稀至500mI.。3.2.2磷酸氢谢溶薇，取5.52R磷酸一氢销(NaH,PO,2H0).溶解丁水中并稀释全200mL3.2.3避盐缓冲溶商：取400mL降单二谢落液、100拉L瞬换二氢钠资及9g期化的，充分溶部并加水至1 C00 m1,(H7.1±C.1)32.4群酸盐明胶级冲被：取0.1g明胺经于1mL磷酸盐短溶液中，期热溶解、3.2.5活性炭：6C日，
3-2.6葡糖：G200。
3.2.了分两齐，（活性块-疏撕管波：取100mR前最晰，洛解于100mL酵酸盐明腔绿液中.器后1g括性炭，电磁挽井1h.于锥形瓶中户4C保存，临测定前用磷酸达-明胶缨冲裁弱希4信。3.2.8甲酵水溶被：20必(V/V)
3.2.9甲杯-磷酸盐-明胶冲蔽，中醇术落液·磷兜盐-明胶缓液<1十4)3.2.10冰乙酸乙醇溶液：冰乙魁十元水乙醇（1十81)。3.2.1虱氧化钠溶液,0.1mal/。
32.12二年甲烷：重莱馅
3.2.132.5-二未基呢哗。
3.2.141,4-效(2-{4\-甲基-5°-苯基唑)1-苯,3.2.15闪怀减，取4x2,3-二率基和0.2g1,1-双2-(1*-甲基-5-莱基博型)]-，游十100Cml二甲苯中，充分混，贵于棕色救内·静胃三日后即可种3.2.16雌二醇.6判甲基B5A抗血清（E-Ab）：上海市内分驱研究所制品或相当者，4C保存。3.2.17雕二醇标准品：纯度SS%（ISASigm=公司提供或相当者）3.2.18准二醇标准储备液：取适量雄二醇标准品，用无水甲醇配成浓度为1mg/mL的标准储备微，出封盘4保弃，
3.2.19雄二醇标准工作被，取一定受的雌二醇标准诺备获，用甲醉-屏醛盘-明胶塑冲被（3.2.3)帮释成10.2，50，10，20，400100的准工作。3.2.20摊二醇氛标记物（\H-E，=38Ci/mmol>，英放化中心Amer9ba:m生产，或相当者，取一是的察液吹率近干.地入解酸益经冲落液（3.2.3)配成70cpm/10L工作能，受4℃保（有效期内使用）。3.2-21净化柱;Scp-uak C小柱，3. 2.22效气.
3.3仅举利设各
3.3.1凌件闪保分析仅。仪器操作条件，机器运行时环境很质为15～80r，相对湿度为39火~80%，确定本仪器点远离所有并放射源。3.3.2较解机，电动
3-3.3低温离心机：本低于8r/mim3.3.4水锅：恒温温度(0二1)℃。3.3.5电磁搅拌器。
3.3.6微注，50~200
3.3.7低钾闪烁瓶Www.bzxZ.net
3.3.8试,75mm×16mm+收变我班*。2
SN0664-1997
转取1试筛（构确至0.1），严洁净的ml具的锥形凝中，加入0.ml.效划化销萨液(3.2.11)及1ml。效甲烷.引盖般抵20 min,干3Stlr/mim离心10mm,弃专上层驾氧化的总并节离凹款甲烷。于哦泄中小加10m一载甲，直复上述举作，含井两次一氟甲烷层，加入5ml.水，报摇1min后于35ccr/min离心12min，弃卡水层.将性收液置4CC水溶中以氢气流以至近十。用2mL事醇水腋游解致密物，将蒂液注人（净化柱，用约10ml，水淋洗，弃去流出滴，用10mL冰Z醛-乙酵溶液（3.2.10)进行洗说，收费10mL洗脱滤于试管中。鼠40心水浴中以氢气吹至近干，临测是前加人1TL甲醇-磷酸盐-明股绥冲液溶解残渣作为最终样液供液体闪烁分折仪测定。3.4.2标准曲线的密司
取1只试管弃编号按下这操作过程制备。1号、2号管作为薄量总T数出。分别入70避微站盐-明腔缓冲湾（3.2.4>和50雕二醇加标记将(3.2.202：，+4静121
3号，4号管作为测母非特异结合宰（M5F)用，分别依次如人3HL瞬酸盐明胶缇冲波（3.2.4)和50m1.雌醇旅标记物（3.2.20>后，4静置13h，加入400产1.分离剂。5号，6号香作为通量持外结合率（R)月，分别依次划入1CC.碎酸±-明胶缓冲胶（3.2.4)、253饿二醇亢血清和5端二醇标记物（2.2.20片，于4置12，入400分剂。7--18号管作标阵落液管用.选10.2C.52.100，200.400Pg/100±L个浓度标准工性满，每个液度用两管。分剂依次加人1C0L标准工作落微、23)L唯二醇抗血流和5UL雌二醇瓶标记物(3.2.20)后,于4℃静置12h,人4叫,分离剂。以上各管静置30nin后以3:00r/min商心2Gmin，吸取每管全部上绩渡，成人闪瓶内，加人10mL闪炼按，静登3h后在滋件闪烁分折夜上测定战射性计数。总1表示标记物在本饮实整中的装度，该法度应和样液中被测物装度长持向一水乎，如偏离太远，宽活当训节试减浓度
按式(1)算B/B.值：
BNSR×109
式中：/，—结合率，路，
H——标生工作微件策康双昏计效均值：B——特异结合双管（即5号、6号管）计数均值；月—等给合双变（邱3号、4号管)控数均值。(
求得月，厅（必）复后，以此为纳坐标，各标准工作领举度为横坐标，车半对效学标抵上绘制标准业线。
3-4.3样夜测定
取2只试编号，于每与试分依次加人10最终样液20雌二醇抗血牌及01期醇减标记物（3.2.2℃）后，了4℃静置131，划人4[.分离剂，以下操作法与制忙标准带线拒国。根据液体内冻分析仪测定的数据，求得/（%）值，再从标准曲线上出样液中赚二醇的依座，3.4.4空白验
使用经脱资案内样，按上考测定步弹述行梁作。3.5结果计算和表述
按式（2>计算试样中端二醇残留含，-V
SN0664—1897
式中：X—试样中推二醇残留含盘，pg/kg；【——从标准曲线上青得样液中惟二醇液度ng/mL】V样液最悠体积，mL
所称试样量R.
性，计族结乐底扣空白值。
测定低限，国收率
本方法的定低限为0.05gg
4.2回收率
牛肉中雄停添刘嵌度及其回收座的实验数据；在0.05g/kg时，回收为84.4元，在0.10/k时.回收事为92.3%：
在50g/kg时，回收率101.2%，
SN06641997
Foreword
This standard was drafted in accordance with the reguiremenis of Gn/T 1.1-19e3 \Directivasfor the wark of atandardixalionUnit IDrnfring and prcsenration of standardsPart 1,General rulesfor drafting standard\and SN/T O0o7—J995\(General rulen fur drufting che atandard methods for thedetermiration of pesticide, vererinary drug Iesidues and biotoxins in comarilities for cxport\ Theuechod of deter ntinatiun of this standerd was drafted on the basis of the fulfil'ed researel wnrk arid byreferring to relevant dnmeslic anit fisreigu lisuretures chrough modificatioa and verificetian. In additiou ,methods of eampling and sample preparacion are alxis xurcified in this standarc.The limit of dererrninarion in this standard is defined on cine baais l ilie turrent inccinaticnelanaximut limits fur estradwl residucs in mcats and meat products aad the zensisivisy oi the nieihoul.Thie standard wae pmponed by and is umler ihr: hargc of the State Admia:atrarion cl Import andExpart Commodity Inepeetion of the Heople's Republic of China.Drafting unit of this astaadardTianjin Import and Export Commodity Iaapectian Bureau uf thePenple's Repullic: of Clhinu.The tuain drafter of this atandard are Liu Ke,Zhung Feugcei.Li Jianying,Q: Yufeng, Yu Run-min
This scandard is n professioral standard prornulgated for the firet time.Notr: Thia Eaglish veraion,a traaslatlon from the Chinese rext,in solely Jor guidance5
Professional Standard of the People's Repuhlic: of ChinaForImportand xportCommodityInspectionMelhol for the deternination of estradiolresidues in meats and meat products for exportRadioimmunoassay
1Seope
SN 0664-1997
This steadaid sgrejfies ahe methods nf sarglink sampke prepsration and determination of estradilul n-silues by radioimmunuassay in eais arut atest praljers far expozt.This sianlard ig aplinable to the duterutinntiun of anirudisl renilun in fruzuu beee for cxpurt.2Samplingndsamplepreparatlon2-1 Inspection .ot
The quanruy of an iuspeeiur lol lemlJ uot lie mure than 2 Stn anekagen.Il churicteristic al ihr turgr wi:hin ihe rame inspectinn lot,such as packing,mark,origin,ptrifin ation and grade.stpuld be tiae same.2.2Qaautityof sampietaken
Nurnber of parkagea
ir. cach inspection lut
26—100
101255
251509
1001—2500
2-3Sr.mpl.ng μrorudir!
Minimllm, puraber l packageste be ti:krn.
A numbzr l pekages speifed in 2.2 are taken al rardom znd openedt one by one. From earh atIzas: one bag xhal. be tiken as a primery gample. In cesc theee are no suall bags invile ri:ch packagc:Dr il thar2 arc imail bars inside eazh [ckug,bu: the cnntest af iiae hag exrerla 2 kg.cul ou. i nutlfrord th: rreul it. euh prekiuge f wt lene thau Jon g with a disinfceted knie. The tata. weight af al?prinsily 5amples shou.d not be less than 2 kg.Mix aad place in a clesn coataine' as tac mixed primarysanigle. lt saould then be sesled,labelad arc aent to the labusarosy in tinsu.2.4 Preparation of tesc saplcPari pf reprtsrrdalive aat:rple ia taker. fmu tlhe mixed primary sample.the edible porlions are hoImegurized ay grinding in a grinder,mixed thoroughly and redeced by quarrering ta at leagr 5od g. Di.vid inro two egua. portiona,p)ace each in ciean contsincr us cha test sample+scel arl lalbel.Approved by 1he Suatr Administratinn nfTonportand Export Cammodity lnspeetion cfthe Peopl's Repablic of Calna ot Aug.15. 1997Implemented[romJan..195E
2.5 Storage of tesr garnple
SN 0664-1997
The teat aample should be storui brlow l (:.Mote: Lr, the course of kampling nnd wample perphration,precputionk must be (aker to hvoid coizlauinztic:n eor oyEaezors wrhich may couse the chnnge of rekidue cnntent.3Method or determination
3.1Principle
The estradiol residucg in the test sample are cxcraeted wicn dicalo:amethauethe excract is evapo-Iated and the residue is leached wilh inethanol solutivti, The solution is cleansl up by EBssing thrdugha Cu,Unlegg oraerwisc gpccified, the rragcnts uscd shocld be enalytically purr.warer\ :g recistil'edwalrr
3.2. 1Diaodi:jm bydrorea phesphate solution: Weikh 3i. 81 x nf disnciun hyd:agen phaaphal(Na,HPO, +12H,O),diasolve and dilute to 500 mL with water.3.2-2 Sodum dibydrogcr. phosphate go:uion: Weigh 5. $2 g oi gocium d:hycroger. psaaphate(NaH,PO, - 2H,Q),lissolve and dilule 1 200 m1. wiih waler.3.2. 3 Phasphate buffer solution, Mix 150 mL of disodium bydrogen phosplate salution, 1on ml. αfscdium dihydrogen phosphate solution and 9 g of sodium chiorice.dissoive tkororghlyand dilute ta1000mLwithwater(pH7.1±0.1).3- 2.4 Phosphate gclatin buffer yolutiou: Wcigh U. 1 g of gelarin,add 10 mL of phogphate buffcr go-lucion and mix well.
3.2.5 Charcoal,$0 mcst.
3.2.6Lexiraa:G20c.
3. 2.7 Scpereting agen: (dextran-charrpel salution),Disse[ve 100 mg of dextren in 10 mL pd phos-plale-gelatin srluion,thea aull f g o[ rhareoul,stir magrrlicully [ur I b,and sture in a lon aL cuuicalJaek at 4 C.Dilute 4 times with phosphategelatin buffe: solutian hefore us-.3.2.BMethanol aqueoussoiution;2oKcy/v).3 2.9Methanol-ghosphate-gelatir buffer splutionimethanol aquepug golurian + phasphategelarisbuffer solution.(1+)
3. 2. 10Acetic aci:l-elhancl solulion :Glat:ia[ acrtit: nrial.m absnlule eihtng! (I+ sg:.3 2- 11 Sodiur hydroxide solution:0. 1 mcl/L.Dichlorometbane:Redistillec
3.2.132,5-iplenylaxazle.
3.2.141,--Bie[2'-(4'-methyl-5'-phenylnxaanle]-henzene.3.2. 15Scintillator: Diszolve 4 k of 2, 5-diphenylox2zole and 0. 2 g of 1. 4-bis [2'-(1'-m2-hyl-3'-phenyloxazhal)-bcnzcae in 1 oo mL of xylere,mix weli and atore ia a browa glass bostle tor ue as-ter 3 deys.
3.2. 16 Eslradial-G-oxine-BSA anliludy (Eg-Ah>,Product or Stunghei Enducrine Instizafe ur cquiv-alent.Store at 'C.
3.2.17Estradiol atandard.Purity 99% (Provided by Sigma dr equivalent).3-2.18 Eseredtol standard stock solution.Prepare an estradiol standard stock solurion of 1 tng/nL inSN 0664-1997
coneenrratian with methanol abaolure),Seal and store at 'C.3.2.19Estrudiol gteadard working solution,Dilute the gtock solution with methanol-phophatehuffer astuinn (3.2.9) 1n preparr al krries of estradiul standord wprking aolucipr.9 of 10,20,50,1C0,200.-ico pg/100 pLin concentratioe.3. 2. 2CT'ritium labelad eatradioE ('H-E,-38 Ci/mmol>:Produce by Amersham Radischemical CenterEuglind,ur eyuivaleni.Take μruper quanucity uf initial solution,biow ro dryneas and add phoaphatebafler salution (s. 2. 3) to prepare a warking anlutinn of 7 oct uprn/lud μL, Scure ut 4'C.3.2.2f Cleanup columa.Sep-pek C. catiridge.3- 2-22Nitrogrn.
3.3apiris ad epren
3. 3- 1 Liguid Scintiliation Analyzez;Operation candicior Amhient Ifnperalnz fnr rpetuting the machin ts .5-30 Cirelaive hcraidiry ia 30%f—gc%:the location of machine rousc be tar from the radiouivu suur
3-3.2 Fslendar rFlectrical.
3. 3.3 Cerlriluge with refrigezator ;not les than $ cod r,mir.3.3.4Water-bath,Theramostatic.(40±1)C3.3.5 Eleetromagrelie stirrer.3.3.6Miuro-zyringe:50200 μL.3. 3.7 Seir.tillacion vial,Low-patagaium3.3.8Trs tubey5 mmX16 mwith s:opper.3.4Prszedure
3.4.1Extraction
Weigh 4 g of thc tcst aample (accurste ro 0.1 g) iato a clean 50 nL cauical flask with etopperAri:G.B l.r andi.am hylraxial-oluilirn (3:2.ll) nnd IUmL of dichloromethane.then stopper andshake for 2c min. Centrifuge at 3oc :/min Ear lo min,liarard tove uyjwr leyer and take out thedica.ormethaae layer, Add 1o mL of dichloromezaanc to the reeidue and repea tlie abhove step, Cum-bine thu cichloromethaac laycrs add 5 mL of wrter rghake for l nin and centrifuge at 3 GGD r/min Ea.10 r n. Tisea-d thr ejum)es layer. Bluw tse extreet to dryncss with a atream of nitrogen at 40'C in aweter-beth, Add 2 mL of methaaal agueaua aalutinn tn dissolve llie rexidue.lajcct the salutan irto aC cart:idge with a raicro-ayringe,rinse with 10 ml bl watec end discard the eFlluier1.Tlien tlule witbIn L of aceric acid-ethanol solution ar.d collec 1o mL of the eluate. Fvupisrule lo dryress wici :atream of niroacn st 4c C ir.a watar-bath. Add I mL of mraanol-phospate-gelatin buffer golotion tnlissaive ilip -wilun heleirr lrigtinirutigu. Tte solutiwn is ready for determiaation with Jiguid scintilla-ticn analyzer as the sample snluticn.3.2.2Plorring the srandard curve.Tas. I8 i1 ruhs t.nd mark wi-h n'imberg. Prepare the standerd curve arrareding to the follawingprocedu:a:
Tubes No. 1 and 2 are used fo: tle determinatinn if whnle eautut (T), Add 7UU rL of phosphare-gclztin bulfer solution and 50 μL of uritium labeled estrodiol separakely,then keep at 4C for 12 h.Tulinn Nr. S and 4 nru usud for the determination of nongpecific binding (NsH), Add separately3cc μl. af phospltate-gelatin buEler aol.alion anl 5) el, of iritium leabeled eatrdiol in scquence,keep at4 C for 12 h-and inally add 400 μuL of separaing akent.Tabes Na. 5 and 6 are used for cthe determiration of specific bindinp (Be>, Add separately IM μLH
SN0664—1997
of phosphace-gelatin buffer olution,200 gL al Eg-Ab and 50 μL f ritiurm lobeled catrediol in se-qucnee,keej aut 4'C for 12 haand finally add 4c μi. of separating agent.Tirhes No. —l8 are used for ztandard solutions, Sir standard wprking splutiong of concentrationsof 10,20,50,100,200 axid 400 pg/100 μL are used for tutermine.tion, Inin twp tnsi-tuhux fur rech cun.centration,rexpeutively add 100 μt. nf standard solution.200 μL of E;-Ab and 50 μL of tritium labeledeatradinl ia seguence,kcep at 4'C fo: 12h,and fiually acd 4c0 yL of se2ereting agte1.For all the tast-tubes,afrer standing far 31h u,t:enLrifuge al 3 500 r/min Eor 20 min.Transfer allthe supernatant in ach tube inta scintllation vial.add 10 mL of geanillatorler stand for 3 h aad de-terrine the counts with liquid acintillation analyzcrWhole count (T) reiresrnts lu cnnrentralion nf laheled substance ia the determiaatior, Thiscuncentrintiom sk:old be at the same levei as tiar of target substance in the sample sclurion. The ccn-ceurration af the test sulution should he adjusted.:f nccegsary.ta avoid tso rtuca duvinlin>r.Accrding to formulati),culeulutethe value R/i:RASR
B-sg100
BB.the bincing rario,y:
Bthe mue.n valun of elupiex robes countiny far each af tle standard wrorkirg so.utior.;D,the mesn valut of specifie binding duplex tubes (i.e. tubes No.5 and 6) couturirg:Nsethe trean value of nonspecifie binding duplex tubes fi, r, tubes Ns, 3 unrl 4] iasnaling-Lsing the valuus ul H/R,(%) iun nrdinate And the concencrations of the staadard working clutianas aksrissa,plot the atandard curre on a gemilog paper.3. 4.3 Determiaation of aample solutior.Takc iw5 umburl n-twlpg.To Facll lesi-n.tn -sdd 10c gl, of fina! sample solstior.200 ul o9E,-Ah and 50 μL of trilium labeled estradiol in sequence,kee2 at 4 for 12 h,then add 4u ,μL f zujarating agent. Proceed an that [or the preparetisn of atandard curc. F-u he alute detetninni: h:y liguidsciatillatiou aunelyzur.clrulate tta, value B/Hu(y:) and find the cpacentration of estradiol in the sam-ple solucion from the etandard curve.3.4.dElsnk test
Lst Ilir sarnple afrr rrruvail af lrrimone ax the blaak sample und determine accord:ng to aboveprncedure.
3.5 Calcuiarion and expreasion of resultCaltuie the uunteri f cstruefiul rriluen i.in test sample according to Iormule. t2)XP
Xthe resiguc concent of eatradiol in ca tes: serple.g/kytc,he rancrr:1ratinn nf traliul in sninple slition pbtained from the standard :urve. ng/rl:V-tae final volume of the seniple salution mL;athe masg of che test sampleig.Nute: The blank valu, sheald be sublracted frcm the zbove seailt cf ca.culatian.4Limil at delermiontinn and rerovery4. 1 Limir of determinalion
SN0664-1997
The limit of determinacion of thig mrthad ia O. o5 pgkg4.2Reeuvery
Aacnrding co the experimented date,the fortifying concentrations of eztradiol in beel and its corre-sponding reroveries are:
3. 05 μg/kg1the Tcrovcry 84- 41% ;1. 10 J-g/kg+the Thivery 92. 3%!3..io μg/kg+therecovery101.2%.10
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