【商检行业标准(SN)】 出口肉及肉制品中苯硫苯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SN0638—1997
出口肉及肉制品中苯硫
苯咪唑残留量检验方法
Method for the determination of fenbendazoleresidues in meats and meat products for export1997-08-15发布
中华人民共和国国家进出口商品检验局1998-01-01实施
SN 0638—1997
本标准是根据GB/T1.1一1993标准化工作导则第1单元，标准的起草与表述规则第1部分：标准编写的基本规定》及SN/T0001-1995出口商品中农药.兽药残留量及生物毒索检验方法标准编写的基本规定》的要求进行编写的，其中测定方法采用了美国农业部安全检查署《化学实验室指南》中的苯硫来咪挫残留量分析方法。但在技术内荐上稍作改进，经验证后，按规定格式要求作了编辑性修改。本标准同时还制定了抽样和制样方法。的。
测定低限是根据国际上对肉及肉制品中苯硫苯咪唑残留量的最高限量及测定方法的灵敏度而制定本标准的附录A为提示的附录。
本标准由中华人民共和国国家进出口商品检验局提出并归口。本标准由中华人民共和国天津进山口商品检验局负资起草。本标准主要起草人：唐翊、修晖、高晓敏。本标准系首次发布的行业标准。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苯硫
苯咪唑残留量检验方法
Method far the determination of fenkiendazoleresidues in meats and meat products for exportSN 0638—1997
本标准规定了出口肉及肉制品中苯硫苯咪唑残留量检验的抽样、制样和液相色谱测定方法。本标准适用于出口猪肉中苯硫苯咪噬残留量的检验。2抽样和制样
2.1检验批
以不超过2500件商品为一检验批。同检验批的商品应具有相同的特征，如包装、标记、产地、规格和等级等。2.2抽样数量bzxZ.net
批量，件
1~～25
26~-100
101~250
251~500
501~1 000
1 001~2 500
2.3抽样方法
最低抽样数，件
按2.2规定的抽样件数随机抽取，逐件开启。从每件中取一袋作为原始样品，其总量不得少于2kg。放入清洁容器内，加封后，标明标记，及时送实验室。如每件中无小包装或有小包装但每袋重录超过2k名者，则可用灭菌后的锋利刀，在抽出的包件中，每件割取不少于100多，混合后置于清洁容器内，作为混合原始样。混合原始样的重量不少于2k。加封后，标明标记，及时送交实验室。2.4试样制备
从所取全部样品中分取出1kg，充分绞碎混匀，分战两等份，装入清洁的容器内密封，作为试样，并标明标记。
2.5试样保存
将试样于一18℃以下冷冻保存。注：在抽样和制样的操作过程中，必须防止样品受到行染或发生残留物含量的变化。中华人民共和国国家进出口商品检验局1997-08-15批准1998-01-01实施
3测定方法
3.1方法提要
SN 0638-: 1997
试样中残留的苯碗苯咪唑用乙酸乙酯提取，用无水硫酸钠脱水.经正已烷和乙醇-盐酸混合溶液分配净化，用配有紫外检测器的高效液相色谱仪检测，外标法定显。3.2试剂和材料
所用试剂除另有规定外均为分析纯,水为蒸馏水。3.2.1 乙酸乙酯。
3.2.2无水乙醇。
3.2.3 正已烷。
3.2.4甲醇：液相色谱用。
3.2. 5 二甲基亚砜。
无水硫酸钠：经650C灼烧4h.置干燥器内备用。碳酸钾溶液:4 mol/I ,将 552. 84 瓦水碳酸钾溶于 1 0C0 mL 水中。3. 2. B盐酸溶液: 0. 2 mol/L,将 16. 5 mL 浓盐酸溶于 1 000 ml 水中。乙醇-盐酸溶液：将66ml.乙醇与33mL盐酸溶液（3.2.8)混合，每两周配次。3.2.9
3.2.10氮水(1+1):取1份滋氨水加1份水。3.2.31磷酸二氢铵缓冲液：0.0imol/1，将[.j5g磷酸二氢铵用950ml水溶解，用氨水（1+1)调市PH 至 7. 0,并定容至 1 000 rrL.3.2.12苯硫莱咪唑标准品：纯度?99%。3.2.13苯硫苯咪唑标准溶液：准确称取适量的苯硫紫咪啦标准品，溶解于二甲基亚砜，配成浓度为1.00m/mL的标准储备液，根据需要再用年醇配成适当液度的标准工作溶液，3.3仪器和设备
3.3.1高效液相色谱仪：配有紫外检测器。3.3.2捣碎机
3.3.3振荡帮。
3.3. 4离心机。
3.3.5涡旋混合器。
微孔过滤膜：c.45um。
3. 3.7玻璃抽滤器。
3. 3. 8 离心管:玻璃,具塞,50 ml.。3.3.9微量注射器：100。
3.4测定步骤
3.4.1提取
称敢试样10g（精确到0.1g）于50m.1.离-心管中，顺序加入19g无水硫酸钠、1mi.碳酸钾溶液及30ml.乙酸乙，盖塞，用手振摇30台，于振荡器上高速振荡10min。于2500r/min离心5min。将上清液通过盛有20g无水硫酸钠的滤纸过滤到另一个50ml.离心管中。再向第一个离心管中加入20mL乙酸乙酯，重复振荡提取，过滤如上述。合并提取液于第二个50mi离心管中。在55C水浴上用缓缓氮气吹去溶剂。
3.4.2净化
向上述离心管中加入20mL正己烷，在涡旋混合器上振荡溶解残渣，再加入20mL乙醉-热酸溶液（3.2.9），充分振摇后，于1000r/min离心2min。充去上层已烷（不要下扰到分界面处的乳化物）。再加入10mL正已烷，重复上述操作，于40℃水浴上用氮气缓缓将下层溶液吹至近干，月2.mL甲醇溶2
解,备 HPLC 测定。
3.4.3测定
3.4.3.1色谱条件
sN 0638-1997
a）色谱柱：RP-Ci柱，YWG，250mmX4.6mm（内径）,10μm或相当者：b）流动相：甲醇-磷酸二氢铵缓冲液（8十2)，配制后经0.45um滤膜抽滤，脱气：c）流速:0.5 ml/min;
d）检测波长：298nm；
e）进样册：20 μLi
f）柱温：25℃。
3.4.3.2色谱测定
根据样液中苯硫苯咪唑的含量情况，选定峰高与样液相近的标准工作溶液。标准工作溶液和样液中苯硫苯咪唑响应值均应在仪器检测线性范用内。标推工作溶液和样液等体积参播进样测定。在上述色谱条件下，苯硫苯咪唑的保留时间约为10min。标准品的色谱图见附录A中图A1。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)计算试样中苯硫苯咪唑残留含量X=hc.V
fg + m
式中：X—试样巾苯硫苯咪唑残留量，mg/kg；h样液中苯硫苯咪唑的峰高，mm;A。标准工作溶液中苯硫苯咪唑的峰高，mm；一标准工作溶波中苯硫苯咪唑的浓度，/mLV
-最终样液的体积mL
—最终样液所代表的试样量·。注：计算结果需扣除空白值。
4测定低限、向收率
4.1测定低限
本方法的测定低限为0.02mg/kg：4.2回收率
回收率的实验数据：
苯硫苯咪唑添加浓度在0.02mg/kg时，回收率为81.4%；苯硫苯咪唑添加浓度在0.10mg/kg时，回收率为82.0%：苯硫苯咪唑添加浓度在0.20mg/kg时.回收率为91.4%，(1)
sn 0638-1997
附录A
(提示的附录）
标准品色谱图
图A1苯硫苯咪唑标准品液相色谱图SN0638—1997
Foreword
This standard was drafted in accordance with the requirements of GB/T 1.1-1993\Directives forthe work af standardization-Unit l:Drafting and presentation of standardsPart l :General rules fordrafting standards\,and SN/T 0001---1995\General rules for drafting the standard methods for thedetermination of pesticide,veterinary drug residues and biotoxins in rommodities for export\, Thisstandard adopts the relevant method for thc determinarion of fenbendazole residues in \Chemistry LabGuidebook\of FSIS,USDA,as thc method of determination,which is technically. equivalent to that ofthe original, butwith slight modlification. After going through verification,this standard was drafted ac cording to the editorial requirement far the stipulated format. In addition, methods of sampling andsainple preparation are also specified in this standard.The limit of determination in this method is defined on thec basis of the curreni international maxi-mum limits for fenbendazole residues in meats and meat products and the sensitivity of the method.Annex A of this standard is an informative annexThis standard was proposed by and is under the charge of the State Administration of Import andExport Commodity Inspection of the People's Republic of China.This standard was drafted by Tianjin Import and Export Cammodity Inspeetion Bureau of thePeople's Republic of China.
The main drafters of this standard are Tang Yi, Tong Hui,Gao Xiaomin,This standard is a prafessional siandard promulgated for the first time.Note: This English version,a translation from the Chinese text,is sulely for guidance.Professional Standard of the People's Republic of Chinafor Import and Export Commodity InspectionMethod for the determination of fenbendazole residues in meats and meat products for exportScope
sN 0638-1997
This standard specifies the methods of sampling,sample preparatiun ant determination of fenber-dazole residues by liquid chromatography in mcats and rmeat prodets for export.This standardl is applicable lo the determination of fenbendazole residues in rork for export.2 Sampling and sample preparation2. 1 Inspection lot
Each inspectior lot should not exceed 2 5oc packages.Thc characteristics of the cargo within the same inspection lot, such as packing,mark,origin,specification-grade etc. should bc the same.2.2 Quantity of sample takenNumber of packages in
each inspectian lot
1—25
26--100
101—250
251—500
501--1 000
1 091-2 500
2.3Sampling procedure
Minimuri number of
packages to be taken
A number of packages spccified in 2. 2 are taken &t random and opened one by one.From cachpackage,at least one bap shal: be taken as primary stmple. The total weight ai all the primary sam.ples should not he less than 2 xg:which shall he placed in a clean contairer ,sealed,labeled and sent 1claboratory in time.
In case the meat pieces are not contained in small hags inside each package,or if there are smallbags inside but the contert of bag exceeds 2 kg,cut out a part from the neat in each package of notless than l00 g with a disinfected sharp krife. Mix the paris of thc mcat as the mixed primary sample.which shall not be less than 2 kg. Place in a clean container,seal,lahcl,and send to the lboratory intine.
2. 4Preparation of test sampleApproved by the State Administration ofImportandExportCommodityInspcetlon ofthePcoplc'sRepublicnfChinaonAug.15.1997Implemented from Jan.1, 1998SV 0638-- 1997
The combined primary sample is reduced to 1 kg and homogenized by blending. The homogenizedsarnple is divided into two equal parts as the test samples which are placed in clean containers ,sealedand labeled.
2. 5Storage of test sample
The test samplc shall be stared below -18'C..Note:In the course of sampling end sample preparation,precantions must be teken to avoid contamination or anyfactors which niay canse the change of residuc conteni.3 Method of detcrmination
3. 1 Principle
The fenbendazole tesidues in the test sample are extracted with ethyl acetate,dehydrated with an-bydrous sodiun sulfate,cleaned up by partitioning with n-hexane and ethanol-hydrochloric acid solu-tion mixture,and determined by HPLC with UV detector using external standard method.3.2 Reagents and materials
Unless otherwisc spccificd,thc reagenis lused should he analylically pure,\water\is distilled water.
3.2, 1 Ethyl acetate.
3.2.2Ethyl alcohal:Anhydrous.3.2.3 n-Hexane.
3. 2. 4 Methanol ;HPI.C gradc.3. 2. 5 Dimethylsuifoxicle.
3. 2. 6 Anhydrous sodiuti sulfate:lgnite at 650'C for 4 h and store in a desiccator.3.2.7Potassium carbonate solution:4 mol/L,dissolve 552.84.g of anhydrous potassium carbonate in1 000 ml, water.
3.2.BHydrochloric acid solution;0.2mol/l.,dilute 15.5 mL, of concenirated hydrochloric acid inI 000 mL of water.
3. 2. 9 Ethanol-hydrochloric acid solution:Combine 66 ml, of ethyl alcohol with 33 mL of hydrochlo.ric acid solulion(3. 2. 8), Prepare freshly every two weeks.3. 2. 10 Ammonium hydroxide solution(1+1).Dilute one volume of concentrated anmmonium hydox-ide solution with one volume of watet.3. 2. 11 Monobasic ammonium hiphosphate buffer: 0. 01 mol/L. Dissplve 1. 15 g of NH,H,PO, in ap-proximately 9s0 ml. of watrr,adjnsi to pH 7. 0 with atnrnoniumn hydroxide solution(1-f 1),and diluteto final valume nf 1 0oo mL.3.2.12 Fcnhendazple standard:Purity≥99%.3. 2. 13 Fenbendazole standard solution; Accurately yreigh a suitable amount of standard fenbenda.zole standard,dissolve in dimethylsulfoxide to prepare a solution of 1. O0 mg/mL in concentration asthe standard stock solution, According to the requitement,prepare a standard working solution of ap-propriate concentration with methanol.3.3 Apparatus and equipment
3. 3. 1 Liquid chromatograph:Equippetl with UV detestor.3.3.2 Blender.
3. 3. 3 Mechanical shaker.
3. 3.4 Centrifuge.
3. 3. 5 Vorrex mixer.
3. 3.6 Membrane filter :0. 45 μm.3. 3. 7 Glass suction device.SN 0638—997
3. 3. 8Centrifuge tube:Glass,with cap,50 ml..3.3. 9 Micro-syringe:100 μL.3.4Procedure
3.4.1 Extraction
Weigh l0 g(accurate to 0. 1 g)of the test sample inito a 50 mL centrifuge tube,add 10 of anhy-draus sodium sulfate,1 ml, of potzssiur carbonate solutiari;and 3c mL of ethyl acerate to the tube insequence.Cap the tuhe and shake vigorously by hand for 30 s,then shake for 10 mir on the mechanicalshaker at high speed. Centrifuge the lube for 5 min at 2 500 r/min. Decant the supernatant thraugn afilter spread with 20 g of anhydrou sodioml sulfate into another 50 mL centrifuge tube,add 20 ml. ofethyl acetate to the first tube again and repeat the above process. Combine the extraci solution into thesecond tube,evaporate aff ethyl acetate under a geratle stream of ritrogen on a 55'C water-bath.3.4.2 Cleanup
Add 20 ml. of n-hexane to the above tube,shake the tuhe on a vortex mixer ta dissolve theresidue. Add 20 nL of the ethanol-hydrochloric: acid solutior(3, 2, 9),shaxe thorouighly. Centrifuge thetube for 2 min at 1 oo0 r/min, Discard +he upper n-hexane phase (zvoid disturbing the emulsicn pre-sent at the interface), Add second 10 mL of n-hexane to the tube and repeat the above process. Blow 1onear dryness under a gentle stream of nitrogen in a 40C water-bath, Dissolve the residue with 2. O mLof methatol and the solution is ready for HPLC aralysis.3.4. 3 Determination
3. 4. 3. 1 LC operating conditions :a) Column;RP-Cie, YWG,250 mmX4. 6 mm(id),10 μm or equivelent :b) Mobile phase;methanol-monobasic armmonium phosphate buffer(8-2).filtered urder varuumthrough 0. 45 μm filter and degassed;c) Flow rate;0. 5 ml/min;
d) Dctector wevelength:298 nm:e) Sanple size:20μl:
[) Column temperature : 25 C,3. 4. 3. 2 1.C determinatic
According to the approximate concentration of ferbendazole in the sample solutiar, select thestandard working solurion with similar peak height to that of the sample solution. The responsrs offenbendazole in the standard working solution ard sample solution should be within the linear range ofthe instrunental detectian. The standard working solution should be randomly injected in-between theinjections of the sample solition of egual volume. Under the above chrcmatogrsphic condition,the re-tention time of fenbendazole is about lo min. For chromatogram of the standard see fig, Al in annexA.
3.4.4 Blank tesl
The operation of the blank test is the aere as that deserihed in the method of determination butwith the ommission of the sample addition,3. 5 Caiculatian and expression of the resultThe calculation of fenbendazole content in the sample is rarried out by LC data processcr or ac.8
cording to the formula(1) :
SN 0638—1997
X --the residue content of fenbendazole in the test samples,mg/kg:h -the peak height of fenbendazole in the sample solutian,mm;h, the peak height of fenbendazole in the standard working solution,mm;-the concentration of fenhendazole in thc standard working solution+μg/ml.:Vthefinal volumeofsamplesolutionmL;m -the correspanding mass of test sample in the final sample solution,g.Note : The blank velue should be subtrected from thc above rcsult of celculatian.4Limit of determination and recovery4. 1 Limit of determination
The limit of determination of this method is 0.02 mg/kg4.2Recovery
（1）
According to the experirnental dala.the fortifying concentrations of fenbendazole and its carresponding recoveries are:
0. 02 mg/kg,the recovery 81. 4%;0. 10 mg/kg,the recovery 82. 0% ;0. 20 mg/kg,the recovery 91. 4%.10
Sn 06361997
Annex A
(informetive)
Chromatogram of the standardo
Fig. Al Licuid chromatogram of fenbcnrlazolc standard
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