【商检行业标准(SN)】 出口肉中丙硫咪唑残留量检验方法

中华人民共和国进出口商品检验行业标准SN 0207 -93
出口肉中丙硫咪唑残留量
检验方法
Method for determination of albcndaznlcresidues inmeat for exprt
1993-06-04发布
中华人民共和国国家进出口商品检验局1993-08-01实施
中华人居共和国进出口商品验行业标准出口肉中丙硫咪唑残留削
检验方法
Method For determinatlon of albentazoleresidues in meat for expurt
1主内容与适用范围
SN 027--93
本标难规定了出口内中丙靠咪唑残国量检龄的拍样，制样和高压箍相仁,谱到定方法，本标准适用于出口猪内中因疏呼唑残留的检验。2抽样和制样
2.1检验批
以不超选2600件一检批，
同检验批的商品应具有相同惩，虾包装，标记、产地、规格利等级等，2.2拍样数量
检验批中的件数
26 ~1:
10:-~25c
251-~ 500
5C- ~-1 CC6
1 CC1.2 5c8
2.3独起法
母心抽样件效
我22规定的抽样件数随机抽收，选件开肩，每件竿少收袋性为娠始乱，原始样品总重不少下2g，放人清洁容器内，加封店，标要标记，及时送实验实.2.4试样制备
从料装原始样品中取出部分有代表性性品，等可含部分放入高连组研严机中换碎均句，分通对，用四分张编分出不生十500，婺人洁净容器内.过封后，标时标记。2.5试样保存
格试样于一18下冷冻保存。
注：在指告和制普过偿十，必所防止性品受到的案或发尘或留物含量的变化。3测定方法
3.1方标提要
2.5均均试样了6NHCI中水解，用碳酸纳调节H8.)用乙融乙酯提取，将两蔬呕坐代谢物和内标反提取到1NIIC1内.丁Sep-Pak柱中净化后，涨缩至下，残链新潜解于流数相中，用具荧光检中华人民共和国国豪进出口商品检验局1993-06-04批准1993-08-01实施
淘器的HPIC进行定盘、
SN 0207-93
3.2试剂和材料
32.15（丙硫酰-1H苯并咪唑-2-胺,标确品。纯度>9%。3.2.2S-(丁炭既）-1HI莱并味型-2-胶：内标。纯度>59%，3.2.3恢盐酸:分析统.
二中亚风（DMSO)经玻剪仪器蒸瘤3.2.4
乙酸乙酯：经股璃设器棘增。
甲醇祭鼓瑞议器热馏，
莱：经玻璃仪器蒸馏。
乙喷经戒璃仅器然询，
乙醇胺，
无水球限的：粉末状+外行纯。
磨酸二双钾，分板纯，
无水群鼓氢二钾：分析统。
3.2. 13水去两手水
氢气纯盘99.%。
3.2.15标准落备游液
3.2.15.1款取10.0mms-（内硫）-2H未并味唑2胺于1001m1.量瓶内用DM5U序解并稀释率到度（100jg/ml）
3.2.15.2欧取10.0mg5-（T硫酰)-1H苯并味唑-2-按于100mL实鼠瓶内，用DMS0焙解井释至线度（10g/n1)。
3.2. 16标准T.作获液
3.2.161移最2.0mL第冬落（3.2.15.1>及8.0mL.DMS5O入-15mz具戒塞需心管内，以满式现匀器混勾，制得2V%/ml.落（3.2.16.1）（2.5试样中加入5C此减，含量相当丁4cc μg/kg>,
3.2.16.2移 5. 0mL上作溶液(3. 2.16.1)及5. 1 mLTMSO人--15mL具玻密离心等内,以病式混勺录混纠.得10g/mL路波（3.2.13.2>（2.58试样中加人50L此警成，套低相当下2c9g3/kg:.
3.2.15.3移取5.0mL作格液（3.2.1e.2)及=.CmLDMS0人一1ral.具效塞离心内，以涡式湿器港句，制保5μg/ml籍液（3.2.15.3)(2.5g试样中:入50μl比游度，含量柑当元100g/kg）3.2.16.4移取1.0mL储签陷液（3.2.15.2)及9.0mLDMSU人—15ml具玻寒高心管内，以涡式混与器促，前得10%/ml.落液（3.2.16.4)<2.5g试详中加人59L此游液，含取相监于200g/kg,
实有标准帮使均应保存于冰箱内，标准生工MSO溶滋中至少可以稳定6个习，医为在此系件下IMSO会发生凝结，所以使用的无要将离心督放于装有水的烧松内，使标准落液重新凝化.以涡式海器混奇。
3.3仪器和设备
3.3.1波柜色游龙配备炎光控测器，3.3.2微量注射器：10μ.5Cl.
3.3.3保护注:CO,PLLE ODS,2mm(内径1×5 :m,3. 3. 4孔过滤器,0. 2 m,0. 4 m滤膜。3.3. 5歧英刻度离心管：具磨塞,15 mL。3.3.6高心机
3.3.7摘碎机。
3.3.8吸秘。
3.3.9pH It.
3.3.10涡式混与器，
3. 3. 11 典空拍滤器。
3.4测是步
3.4.1提取和净化
S20793
3.4.1.1取试样代表生部位，以将确机进行充分约化．划化后的试样在分析前一自冷冻保在。3.4.1.2将一六皮试的堵杯胃于天平上.虑杯中效50mL裁离心管（米加塞），用管尖吸移件取样，称助2.5士0.05名均勾试群，图于高心算低。如书分别制各四份空白组织试样，及一份对照试详：三份乘加标准的试样，在对照试样中加I入InL纯DMSO。在疑有丙惊咪唑州物的组织试择中加人50μDMS0.在宣白殖缺试样和疑有丙硫映唑的组织试样中分别加人5UrL工作落液（3.2.16.4)。熟后，在已加人50L工作落摘（5.2.16.4)的三个空自组织试样中分别加入5CL工作溶液（3.2.16.1)、(3.2.16.2).(3.2.16.3)。
注：应游录加落滋女十5管内叫好院改均与让样，以左下步加人CI时南保有足够的落满3.4.1.3存3.4.1.2制备的每试择中加入2.Gm[.6NHC1，益好每支管，然忌将试管（用另一试管架固定，以防整子炎然出>放到预先调至110士4℃的烘箱内。3.4.1.4在7五后，把试以113土4的烘整中依出，将试摔带放十下冰或冰上对5min，冷至室混，
3.4.1.5向每个试挡中加入2. 0mL水,然后以误式混句能源试样，月pH让监测rH直。慢慢加入足量（约1.23g)的炭酸的.加入时间约等4~Emin，以免起泡变结决，调节2H王8~9.5(将活净的pH也极直接成入试样中函定：记录H值后，在离心算内壁上轻轻接触去掉电极上的限演，将驾个电报从武样管中收出，用水冲欲，接于后再测意下一个试样），注：幸阻或疾近室湖的欧样，加人策酸铺书策校冷试那择不会产生内澳，固此·加人渊酸销了可以快些。34.1.6向每个试样内加人15ml-2醛2酯，登塞子，放于试管架上，标5min，筋后取下案于，于约44ocor/min等试样离心5min。3.4.1.7用吸移管将乙践乙陷提取液转人另一5:1l.破璃离心首内：应尽最转体光全。法烹不要带造水相。
3.4.1.8益复频作3.4.上.和3.4.1.7步照，再次提取每个试样，把相应的提取拨个并于各白的5mL变璃两心管内。
3.4.1.向每个会并均Z.酸Z酯提版极中加入4.0mL1VH2签上窕于，平放于试等架上.报据5min，取下密子，然后于4roo1/min左右离心5min3.4.1.10尽基完余吸去乙酸乙弱相.注意不要诺走水相。左35士5求游上两去水籍液上的乙龄酯（1mL)，《必要时.可在比步骤倍止分析探作，雨放置过。）3.4.1.11以涡式混句器将每一试样泻匀同时性慢加人足量（终0.3g）残酸钠，加入时约需1-2min,以免起泡或结块。用H计监测rH值，每个试样的pH值应为R~9.i.3.4.1.12向每个试样中人201L革，益上案，平较于试管架上报搭5in，收下家了，于4000Imin充将试样离心5mir，
3.4.1.13尽量完全吸去甲求明，坐意不要带走水相，坐35士拍水溶上藻去水对上效留的甲兼（1.），（必费的可杂此步录停正分析操作，前放互过夜。）3.4.1.14对下轻-个试，拒对产将一10mL我璃注射器（圆栏形推做塞要取下>用光子周定环形架F。注射器的群氏接头前端连接SEFPAKC13忙位.3.4.1.15软每支柱用2mI甲醇预洗.而后用2mL0.M磷酸钾缓冷漆准（pH8.0)洗渐，加人每种试SN 0207--93
剂后，经过安装了于注射器顶部的橡皮塞级缓通入氮气，利用氢气压力使试剂穿过柱体。当氮气并始出柱时即停气，需气流迷约1-2mL/mia奔去脱液。3.4.1.16从步骤3.4.1.13中得到的每一试样水相转入到先洗好的各坐射得柱系境内，用步魔3.4.1.1-的方法、应用效气使溶液流经柱体洗提，然后各取每一试样替水洗设1mL（经需式混勾器混}使用巴氏吸移胃采用类假方法将水选波分别转入各江射器性最统内，弃去洗脱滤。3.4.1.17再收每个试机管水沈浪1mL（经弱式据守器混)，分别转人各注射靠忙系统内，按步赖3.4..15的书法应用熟气使此水洗夜流经控体选提，齐去洗脱滤。3.4.1.18向每一注射器性系统内分别加入2ml.甲萃，按步聚5.4.1.15的方供用氢气快甲革流经偿体洗提,弃去洗脱狱。
3.4.1.19在每-注射需需统下而分别效置一15mT.商心誉，向每个桂系统内加人2mL乙酸么按步聚3.4.1-15的方法用氮气使乙峻乙髓潮经址体进行洗股，在这部分洗脱核中含有待测北台物。3.4.1.20向步骤3.4.1.19得到的每份选悦商中分系证入约.5mL申，以满式混约器勺，以得到均的蒂液，
3.4.1.21于35土5的水浴上在干燥效气下将步双3.4.1.20得到的选说浪浓瞻至干3.4.1-22举干后，加人C.5mL水-单落被（70十30），以涡式混勺器混勺.重新溶解残适。3.4.1.23/30m后，将每个单新解的试详知入微所过透装量内采用0.2m生行继承滤膜过滤，然后将每个过滤器及试样提取液于4ccor/min左右离心5--10min。3.4.1.24将够液转入：5ml.具塞我离心管内，用水中醇（75+30）节每一泄被件积为0.5mL，使用群烷化暨有十将提取液放靠于管约底部，以离式混勾器混勺后移取.1mL供HLC分析。3.4.2测
3.4.2.1液色谱茶件
a.色柱：pPnndapakC18桂，30crn×3.9rum内径b.饿动档.68%0.02MKH.PO,0.(1M.乙醇胺(200mLU.1MKH,PO1.05g=乙醉胺加水L）
20中：
12%艺晴。
该溶液当以新配,使用前用I1.45Tn滤膜逆滤并用克些系统抛气c.流速1.8ml./min
d.温文室道、
3.4.2.2色谱湖定
先将融发波元和发射波长分别测在393nm和32cnm，狭键完度：3nm。设置合理的基线减敏没，注入适些成拦（20北，然后观察意和所标响尚降时强质，若需更虾的灵数度，圳进行如下求骤，在观案检测器喷收的应时，m以内调激发波长，以减小本底值，如上再次邀样，确定微测化合物的信号响应无零烂加，接此序继续选择联佳激发波长和发射波长，直至得到合适的分所信操比(2/n=25/1).
在上系计下，其保留时间：内统峡唑代谢物约4.3m.内标约7.0min。3.4.3结果计第
若改有乳分仪，则可以用峰商值和峰高比来代下面所讨的而积环峰面界比。对于非个试片提度设，HPLC测危所得再殖迷唑代物转晓而识除以内标转峰面积，得到蜂面积比
峰面职比丙味代谢激蜂面积
内标峰骨积
SN0207-93
在直角坐标上，以样面积比为缺坐标，以对照试样加人标的严/值为模垒标作工作前线，摘曲践得国归力程。由回归方程从未知试样的峰面奥比按下式直接计算丙研咪唑代谢物的g/kB值，X
式中，—-得到试样中闲硫咪碳代谢物的浓度，/g1一待测试样探取城的峰面积比
b——·回归方的极距：
切一一回归方程的斜本，
4方法的测定低限和回收率
4. 1测定低限：25 pg/xg-
4.2回收率
回收率的实验数据：两唑代谢物浓度范005~0.20m2/8时.回收率为86.9%~-03.25%，附加说明：
本标准比中华人民共和国限家逆出口商品检验局提出。本标准由中华人民共和国制北进出口商品检峻局负费起卓，本标准主要起蓝人语受杰，
主考文献
FSTS Chem.Lab.Gutrdebeok 5.c34.198Professional Standard of the People's Repabllc of Chinafor Import and Export Commodity InspectionMethod for delermination of alhendazoleresidues in meat for exprt
TScopeand fteld or applicatlonSN 0207-93
Thi: siandard spezifies the methods of sampling,sample preparation and determinatian by liguidchromatography of albendazole residues in tueet for export.Thig 3tar.dard is upplicable tu dcterminacion of albcnaluzole residuex in mat For rxport.2Samplingandsamplepreparation2.1 Inspection lat
The quantity of ar. inaucetiwn lot shorld nol te morr than 2 Snu packegex. The chaunucterislies ofihe cargn within the sarse inspection lot.such as packing,tuark,origin specification and grade, zhouldbe the same.
2.2 Quantty af sample takeu
Nunber ol package3
in eachinspectiun lot
1—25此内容来自标准下载网
25-100
101—250
251—500
591—1 c5
10125c0
Minmumnumberof
packc.ges to be taken
2.3 Sampling prnedure
A tumber cf parkages specihed in 2.2 ere taken at zandou ani openel ae by ouc.Frorn cach atleast one bag sheli be taken as s primary ample.The turel weight of at pimary Samyles shouial not hrlcss thar 2 kgs whish shull be plaued in a rles uuntainer,sealed.labeled and sent ta laboratnry in time,2.4 Preparatina sr1est sanplePart of rcpresentative saruple is taken Irom esch bag of the primary sample aad thc ed:blc portionQE which is hamogenized by grindirg in a meat grindcr. The hpiaogeniwnil sumple is thuroughly mixtu!ind zelured u 5Uu g ly re'aurturiag.Thr test kanuple is plaped in clean contaizer which is sealed and la-beled.
Appraved by the State Administration ufImport and Exporl commudity Iaspertion orthe People's Repuilic of Chiua on Jun.4.19936
Impleniented Fran Aug.1.19932. 5Srorage pl sample
SN 0207—93
The test samrle shoutd be atorerl at -L8c..Note, Io tir couirar nf saayling nnd aample prepatatinnpreraution must be takea eo avoid coneaminatior.or ar.yfactora w'nirh may rainxr che change Df reaidur. rontenr.3,Methnd of determinatinn
3.1Principle
2. 5 g homogenized sample hydrolysed in 6 N HCl.adjusted n pH3. wilh xdim urboratanc extracted with ethyl acetate, The albendarole marker and intornal standard are back-extrarred intn1 N HCl. The agueaus phage ig applied to u Sup Pek Ci cartridge where the compounds ol ir.terest areretuined and tluted with ellhyl acetate. The ethyl acetale extract is thcn corcenatcd to dryness. Theregidue ie Teconstituted in mobile ph.ase.filtered and quantificatian perforiued by HPLC using fluorcs-cence detection.
3.2Reagents and meterials
3.2.1 5-(Propylalforyl)-1 H henazimidaaml-2-aininnphlurulurd substance-qurity≥99%3. 2.25-(Dutylsulfonyl)-1 11 benzimidazn]-8-amineinterna) siandard.qurity>9%.3.2,3Concenrrated hydrochloric acid:A.R.,3.2. 4 Dianethylsuifuxide 3. 2. 9Diel hanolarrine.
Sodium carbonate:anhydzous powder,A.R.3.2.10
Potassium pbosphate monobasic:A.R..Purussium ghaaphate dibeaic anhydrous,A. R.3.2.13Water:deinric.
3.2.14Nitrogen:purity9.g%.
3.2.15 StunGard stock aolutionz3.2.15. 1 Into a liJ r.L voiurnetric flask,weigh 10. 0 mg of 5-(Pro2ylgulfonyl)-1 H benzimifazo.-2-sraine analytical stand:s rd. Dissolve nad iake to mark wich DMSO(1UU μg/mL).3. 2. 15. 2Iata 100 mT, valumetric flank,weiglh 10. n ng sf E-{Butylsulfunyl) 1 H benziridazul 2arniae anelyinil standard. Dessolve aad take to mark with DMSU(1cO μe/n.L.).3.2.16 Siandards working aoiutions3. 2.16. 1 Pipet 2. 0 mT. of stock golution(3. 2. 15. 1)and 8. 0 mL eI DMSO inzo 2 15 rnl glaas *l:2-pered centrifug\ tube nd vortex mix,to produce a 2G μg/ml. snl.lirn (3. 2. It. I1(5 eL fo-titiud t:2. 5 g of gatiple is eijpuiveleat to 400 μg/kg).3.2.16.2Pipet 5.0 mL uf worxing atandard<3.2.16.1)anc 5.0mL of DMSU into a 15mLgiassstoppered ceatrifuge tube anil vurtex mix ,to produce n 10 μg/mL solution(3. 2. 16. 23(50 μl, tarifiedto 2. 5 g of sample is equivalent to 200 jrg/kg).3.2,15.3Pipet 5.Uml af working tandard<3.2.16.2)and 5. mT.ni DMS0 inti a 15 ruL giaxgtoppered cent rilage tuhn und vorrex, mixto produce a 5 gg/mL solution(3, 2. 16, 3)(50 μL forcified ta2. s g of mmple is equivalent ta lao pg/kg).SN 0207—93
3. 2. 16. 4 Pipet 1. 0 mL of stock aolutiun(3. 2. 15. 2)and 9. U mL uf DMSO iutu B 15 nL gins stopPered ceatrifugn tubr and viorlex nsix,ln produce a 10 y/ml. solution(3.2.15.4)(Go μI. fortified ln2. 5 g of manple is equivalent to 200 μg/kg).All standard solutione should be gtored in a refrigtrarer. Srandard solutions are stable in [wisQIsr a minimur of gix mwnths. Bcrausc DMsO solidifies under these ruaditions, it is nccegsary to reliquify che staadard soluliurs by aluring the lubres in a leaket of lukewarm warer and vnrlex mix thesoluligs prior to use.
3.3Apparatus and equipment
3. 3. 11.iquid chrametugrapn wiih fluur'srcnc xpectrorihli>meter.3.3.2 Miero-yrange:1G μL.5 +1.3. 3.3 Gruard cclumn;CO,FELL UDS,2 mm IDX 5 cm.3. 3. 4 Filters:0. 2 μm,0. 45 μm3.3.5Clagggraduered centrifugc tuber15rrL3.3.6Cetrifuge.
3. 3.7 Blender.
3.3. 8 Fipetter.
3. 3. 9 pH mctrr-
3. 3. 1D Variex inixer.
3. 3.11Vacuum filter.
3. 4Procedur
3.4. 1Extractton znd ckeat: uj3. 4. l. 1 Represeat:.live xrrlians of saunple are tharonghly homogenized in a blender un:il no visiblepiecee remsis,Jnavagenates should be kept frozen at all tines until analyzed.3. 4.1.2lnta the boetora of 50 nL glass centrifsae tube (unstappared),supported in bwakur utlon bslaace,weigh 2. b ± D. 05 g of homugerized st.mples using u wirle lip dispnaable piper, Prepsrfuur lack 1isane xanipiertuin fnr cn:alrol asd threr for Fortification- Add 100 pl, of pure DMsO to becoatrel. Add SC μL of DMSU to the tiasuea suapeeted of cuntaining ulhendezulr residue. Add 54 μlL ufworking 2landard solution(3. 2.16.4)tu cuch uf the remaining three biank tissue aamplex aud the tisguc sanples suspgctud of jntaining alberufazale renidue- Add S0 μL of workiag standard bolution.[3. 2. 15. 11(3. 2. 16. 2)t3. 2. 16. 3)ta separate biank tissue aanples frora the set of three already fortified witli warking atandard zolution(3. 2. 16. 4).Nuta : Fu:-ilicat:nz uolutiurie shoulJ be added to the 5o ml, tube just above tbe homogenate cc iosure xolution w ne?the HCl is added ia the aext atep.3. 4. 1. 3 Add 2. 5 mL uf N HCl tu ea.l sanuale prepared in srep3. 4. 1. 2.stnpper cach tube.placeilit. aanaple Iuhrs(cuvereal wit't annries tuhr rack to prevent stoppers fram popping ?in an over. presetto 110-t4'..
3. 4. T. 4 Afte: I h remove the samples frorm the 12u±4C uveu and aliow ihe tlea to ccal to I5auttempaurature ver dry ice pr ice fur approxinately 5 min.3. 2. 1. 5 Add 2. t ml. nl waler 1n ach sample, Wile vortex mixing the sanple,gloaly add cnoughSN 0207—93
kimwige befort: uaanitoring che next sarmple].Nntr: nmples at ar es
sdded feater.
ture ala not fonm na much as cole.nr srmples,Tberetore.the Na,CO,an,be3.4.1.Add 15 ml. af ethyl abetate to each sample and stopper the tubes.Sluake the umple5,supPo:ted En + reek in a horizontitl posirion for 5 min. Kemoue the sioppers &hd rvutriluge the. s.inples atapproximately4 toa /iet fur 5 min.3. 4.1.7 Trangfer as much as panible of the nlhyl acetate xtrutlk 1n eparete 50 nsL giass cenerilugetubes using a disposable pipet taking care pct to transfet sny of the agueous plase.3.4. 1. 8 Re exiract curh xample a suuur umc by repeang stepa 3.4. 1. 6 and 3. 4. 1. 7 and poolingthe approariale ekeracte in th rexarrlive il mL glhs.s eeurrituge tubes.3. 4. 1.9 Add 4. G mL of 1 N I1Cl to eath ethyl acetate po.rdl ektraet arud stopur ihe tubeg. Shakgthe sample3 supported in a rack in a horizontal position for G minutes. Retanvr alie stopairs unel certIriiuge the sampleg at app:sxinietely 4 coy y/min for 5 mintes.3.4.i. 10Remove un turh ms ousiblo ni the cthyl gcezarc phase by aapirxtion,taking care mGt ta retnve any cf che acgucnus phase.Fvaporatr l rernaiuing rchyl cetate(l mL>From the aqueousphase in the waterbarh a: 3s L 5 C. (1f neceesary the hktracticr. can le slapped a: this puinlovernighe)
3. 4.1.11 While vurinx mixirg cach simale.add cnongh tapproximarely 0. 33 g)of aooium carbonareslouly(appraxinateiy 1-2 mi:ures to avcid faming and/et txkirg ). Each samplu shu:rld be az pHy-9. 5 when monitared with a pI meter.3. 4.1. 12 Add 20 mL cf toluene ta each satnple arc opper rabos. Shake the 5amples 5uparte:l in aTack in a hnrizontol pusition.fur 5 ninuns.Remove the stopperg and ceneniduge the gamnples at approxinately 4000 r/i.[ni 5 min,uts.J.4.1.13Renwve as much as pcssisle cf cle Iclurne phane by axpiration te.king taire nuc ta removeany of the ugueous phase.Evuparate the tehaining toluene(.l ml)Eron abnve the aqueuu phaise inthe walrrhath a: $5=B C. (lf uecessery che eatraction can bp stopped at this Pcint overnigh:).3. 4. T. 14Attach a Sea-Pak Cle tr-eitgr to thv Jecr aip of a 1r mL giass syringc scesred ic a ringSend writh u elamp for each sampleicylinder harrel ersveel).3. 4. 1. 15 Prewash eath castridge with 2 tui, nf methanol fallnwed by 2 ml. nf 0. 2 M potasxiuni pluxphate buffer(pHe, o). Each reagsnt is transported through the cartridge by means of genile nitropenpressure applied theaugh nbser atopper fitted into the tup uf the ayring alre addition bl the respec-tive Ieagene.The aitrogen prexhur in tetazaverl witea r:irrigce. begins tl exi: the curridge. Flou rateapprox.1--2 mL/min.Drscard eluatee.3. 4.1. 16 Traaxler thu uuccus phase fron eacts zamp'e from step 3. 4.1. 13 ta t separate prewashedsyringe cartridge unit. Elute this phesc Eren the cartricge using nutrogcn as in srep 3. 4. 1. L1 thentransFera l nLwarer waah ofeuti sinpirtube(veriey-n.ixed'cc thc respccte saringe-carrtdgs unitin 2 grziler mdaner using the respective paswur pipet, Discuridll s.uztes.3.4. 1.17 Aco an andeilionut 1 mL warer wash of czch sampl tube(vortex-mixed)to tl:e rtsaet:tivesyrine-cailridgc unit and elulr using nitrogen xs in step 3. 4, l. .5. Discard elaates.3. 4. T. T8Add 2 ml. of tutaene n aarit syringu-nrtringe unit ynd clute usieg nstragen ies in srep3. 4. 1. 15. Distirt eluates.3. 4. 1. 19Place a 15 mL glass centrifuge tubr hewarlh eacin syrirge-irtidgc ucit. Arir 2 mi. of cthylazetate tn erl urit aad clute using nitrogen as ia step 3. 4. J.15.This trartin: rnntains thr remipcundof nteres:
SN 0207—93
3.4.1.20 Add abouc 0. 5 ml of methanol to tach eluat froru step 3.4.1.19 and vorret mix to obraina hamogeneous solution.
3.4. 1.21 Cancentrate the eluatea from step 3. 4. 1. 20 cu dryncus under lry niirogen in an waterhth35±5℃.
3. 4. 1.22 After reachiuw dryaess aid 0. 5 mL of the water/methnolt70/3)solurion and vortex mix1n rebnngticute the residuc
3. 4. 1. 23 Afte: 30 minutes app'y each of thr recanstituced samples in a scparace birunaly tical system fi.ter urit filtcrced wich an 0. 2 μm rrgenrrulel cellulnar. filitr. Cent.ifuge rarch Elter unic cantaininksampletkirar:dluppruximately4o00r/minfor5—10minutes,3 4.1.24Transfer the filtrates to se7arate 15 mL glasa stoppered centrifuge tubes and adjust thevolume of each filtrates to D. 5 mL with warer/methanol(70/30), Use of a silanized rube is helpful tocoaceutrate the extraet residuc to the very bottom of the tebe. After vortex nixing temiove O, I mil furHPLC anelysis.
3. 4. 2Delerminalit
3.4.2.1Conditions of [IPLC
a,Columm.so emx9.D mmI.D.μBondeyak C18.b.Mcbi.: ghase;65X 1.02 M KH, PO, 0.01 M iachannlanine(230 mI, 0.1M KH,PU,+l.05 dicthaaoiariue tukea lu l lirar wil water),B0%methanalp
12%acetonitrile
This aaluuon is prepared deily+iltered through 0, 45 μra filter and lagasnit under vacuum priora e.
c.Flsw rate...B ml./nir.
Temperature.Ccluma,mobilephase arddeteciortemzerature=ambicnt.d.
3. 4. 2. 2 Determination of HPLCIniszlly gzt the excirazion urt esnission wavelength6 at So0 nm and 320 ntt-respectively with sli- width ofslb ar (ur lioth, Inject an appropriate olunie of sample(2D pL)and oaserve the iatensitysl the resfuonse Inr both standard and internal standard with a reasonable baselire sensitiviry gctting.lr=cditinnsl sensitivity is zequired proceed sa foliows,While observing tae detector absorpcion rusjonseradjust the exsitarinn wiveleagrh ta sg5 nm toTedace the zero wppresaion. Rcinjcct the sin harruple ias abote aml derernine if the signal respansesfor the comjuunds of irterrni im:rrine. Cnntinje this prncedure of optimiziag beth the hxeicetion and e-ninion wav+lengihx lu ghtain an adequate zignal to nojse ratio for the analygig (s/n 25/J).rcer the above candicion,the retention rimealbeadazolc markcr apuruximetely 4. 3 miautes ,interna: staads:d approximaleiy 7.Uminates.3.4.3 Caleulazion ane exirexeiun uf nlr[f an iniegrutor is un.ivail:lile -aeak height nteaaurements and peak heighr ratia duta can he substi-Tulril fn: pezk area anit peak area rao cata in the foilowing discuggian.Dixide rhe peak area of the albendatcle marker by the pek areu of lh irerna. standard frcm thcIIPLC data dor each sarple extraer,to obtain peak Brea zatins.Area nf slbendazole uuarkcr peakPeak area ratio=
Ares of interuu: standard peak16
SN0207-93
Construet an aralytica curve by plorngton lincat axcs,thec peak ares ratio versus the respectiveag/kg value for coul, fortified conitrol xanple. eing the regresion erjutation for he enalytieal curve,direcily taleulate the μg/kg level of albendazole narker in the unknawn samples frorn their Peak arearatios using the following equation;X.-5
wlhrre:
concentration inzuspectsamale.g/kg:Y-peak areeretis of suspcct sample cxtracr;b—-inerrepi framregreaion eujuatinntstope from regression equation.4 Limil of determinatinn and recavery4.1Limit of deterainatiui;25μg/kg4.2Recovery
Aesordng to the eapcrimcncel dasa,when -he concentracion of aibendazole merkcr is in -he tangcof n.05—d.20 mg/kg.1h teravery is86.9%—93.25yAddiilnnalexplanatinas
This etandard was proposed bp the State Administration of Impprt and Expart Comedi-y Ir.spection of tlhe Peopla's Repsbl:c of China.This stendard was drafted by Habei lmpurt and Export Comnodity Insgeetion Bureau of che Pcople'a Republie ne Chinan.
This standard was nainly dralted by Shao Jun'ieRefernces
FSIS Chem., I.ah, uilrborik 5. 34-1388.Yote: Tis E:iglisit vexinn,s trnna!nticn frnr. thr 4'zinear -r.is solnly ier Rn.dnnrr11
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