1、中华人民共和国出入境检验检疫行业标准SN/T 1628-2005 进出口肉及肉制品中氯氧破柳股残留量测定方法高效液相色谱法Determination of closantel residues in meat and meat products for import and export-一-HPLC2005-08-18发布060608000106 2006-02-01实施中华人民共和国发布国家质量监督检验检疫总局前言本标准的附录A为资料性附录。本标准由国家认证认可监督管理委员会提出井归口。本标准起草单位:中华人民共和国河北出入境检验检疫局。本标准主要起草人:王凤池、马振栋、艾连峰、窦彩云。本
2、标准系首次发布的出入境检验检疫行业标准。SN/T 1628-2005 I 1 范围进出口肉及肉制品中氯氨破柳肢残留量测定方法高效液相色谱法本标准规定了肉及肉制品中氯氧腆柳胶残留量的高效液相色谱测定方法。本标准适用于牛肉、羊肉中氯氯腆柳股残留量的测定。2 测定方法2. 1 方法提要SN/T 1628-2005 试样中的氯氧腆柳胶残留用乙睛提取，经正己皖-乙睛液液分配净化，蒸干后用乙睛定容，用配有荧光检测器的高效液相色谱仪测定，外标法定量。2.2 试剂和材料除另有规定外，所用试剂均为分析纯，水为超纯水。2.2. 1 乙睛:HPLC级，用正己烧饱和。2.2.2 甲醇。2.2.3 三乙牍。2.2.4
3、磷酸z优级纯。2.2.5 元水硫酸纳:6500C灼烧4b，贮于密闭容器中备用。2.2.6 正己皖:用乙睛饱和。2.2.7 0.45m滤膜。2.2.8 氯氧腆柳肢标准品:纯度二三98.0%。2.2.9 标准储备液:准确称取10mgC精确至0.1mg)氯氧腆柳胶标准品，用乙脯榕解，转移至20mL 容量瓶中，用乙睛定容至刻度。此榕液浓度为0.500mg/mL, 1 oC 4 oC下保存，保存期为1年。2.2.10 标准中间液:吸取1.00 mL标准储备液，用乙腊定容至50mL容量瓶中。浓度为10.0g/mL， 1 oC 4 oC下保存，保存期为3个月。2.2. 11 标准工作液:根据需要，吸取一定量
4、的标准中间液，用乙睛稀释成标准工作液，现用现配。2.3 仪器和设备2.3.1 高效液相色谱仪z配有荧光检测器。2.3.2 超声波清洗器。2.3.3 旋转蒸发器。2.3.4 氮吹仪。2.3.5 酸度计。2.4 测定步骤2.4. 1 提取称取5gC精确至0.01g)绞碎、混匀后的实验室样品于100mL具塞磨口锥形瓶中，加入50.0mL 饱和正己烧的乙睛和约10g无水硫酸锅。盖紧塞子后，用力振摇，使样品分散开，然后在超声披清洗器上超声提取10mino将提取液用撞纸过滤到150mL分液漏斗中。2.4.2 净化向盛有滤液的分液漏斗中加入40mL饱和乙睛的正己皖，盖紧塞子，轻轻摇动数下后放气，然后振SN/
5、T 1628-2005 摇约2min.然后打开塞子，静置约10min。将适量下层榕液放出到一个锥形瓶中，准确移取10mL.用旋转蒸发器或氮吹仪500C下蒸干，用乙腊定容至1.0 mL.混匀后超声约2min。溶液过0.45m膜后供液相色谱测定。2.4.3 测定2.4.3. 1 灌相色谱条件a) 色谱柱:C18柱或相当者，造用于pH=2.5的流动相.150mmX 4.6 mm(内径)填料颗粒直径5m。b) 流动相:乙腊-水(75+25)+0.05%三乙肢，在酸度计测量下，用H3P04调至pH=2.50c) 流动相流速:1.0mL/min。d) 荧光检测器波长:EX:335nm.EM:510 nm。
6、e) 进样量:20L。2.4.3.2 色谱测定根据试样中被测物的含量情况，选取响应值相近的标准工作液一起进行色谱分析。标准工作液和待测样液中氯氧腆柳胶的响应值均应在仪器线性响应范围内。对标准工作液和样液等体积参插进样测定。上述色谱条件下，氯氟腆柳胶的保留时间为17.6min.标准品色谱图参见附录A。2.4.4 空白实验除不称取试样外，均按上述测定步骤进行。2.4.5 结果计算和表述用色谱数据处理软件中的外标法，或按式(1)计算试样中氯靠在腆柳腊的残留量:式中zX一(A- Ao) C V -As. m X一-试样中氯氧腆柳胶的残留量，单位为毫克每千克(mg/kg); A一-样液氯氧腆柳脏的色谱峰
7、面积，单位为平方毫米(mm2); Ao -空白实验氯氧腆柳肢的色谱峰面积，单位为平方毫米(mm2); C一-标准工作液中氯氨腆柳胶的浓度，单位为微克每毫升(g/mU;V一-样液最终定容体积，单位为毫升(mU;As -标准工作液氯氧腆柳胶的色谱峰面积，单位为平方毫米(mm2); m一-最终样液代表的试样量，单位为克(g)。3 测定低限、回收率3. 1 测定低限本方法的测定低限为:0.1mg/kg。3.2 回收率3.2. 1 牛肉试样中回收率的实验数据添加水平为0.1mg/kg、0.5mg/kg、1.0 mg/同时，回收率在86.2%92. 0%之间。3.2.2 羊肉试样申回收率的实验数据添加水平
8、为0.1mg/kg、0.5mg/kg、1.0 mg/同时，回收率在84.9%95. 0%之间。2 . ( 1 ) SN/T 1628一2005附录资料性附录)氯佩确柳肢标准晶渡相色谱固A 沓暴警回事国喃60 40 20 。gdNH臼rrun 18 16 14 12 氯佩确柳腰标准晶液相色谱困10 8 6 固A.14 2 20 0 3 SN/T 1628-2005 Foreword Annex A is an informative annex. This standard was proposed by and is under the charge of National Regulator
9、y Commission for Certi fication and Accreditation. This standard was drafted bHebei Entry-Exit Inspection and Ouarantine Bureau of the Peoples Re public of China. The main drafters of this standard are Wang Fengchi , Ma Zhendong , Ai Lianfeng and Dou Caiyun. This standard is an Entry-Exit Inspection
10、 and Ouarantine professional standard published for the first time. 4 SN/T 1628-2005 Determination of closantel residues in meat and meat products for import and export HPLC 1 Scope The standard specifies the determination method of closantel residues in meat and meat products by HPLC. This standard
11、 is applicable to the determination of closantel residues in mutton and beef. 2 Method of determination 2. 1 Abstract of the method A sample is extracted with acetonitrile. The extraction is evaporated to dryness under nitrogen at 50t after purified by liquid-liquid extraction of hexane-acetonitrile
12、. Residues after drying are dis solved with 1.0 mL acetonitrile and 20I are injected to HPLC with fluorescence detector. and quan tified with external standard method. 2. 2 Reagents and mater ials AII reagents used in this method are A. R. and water is deionized water unless specifically noted. 2.2.
13、1 Acetonitrile: HPLC grade. Saturated by n-Hexane. 2. 2. 2 Mechanol. 2.2.3 Triethylamine. 2.2.4 Phosphorous acid: GR grade. 2.2.5 Anhdrous sodium sulfate: Ignite at 650 t for 4 h. and keep in a tightly closed container af ter cooling. 2.2.6 n-Hexane Saturated by acetonitrile. 2.2.7 0.45m filter. 2.
14、2. 8 Standard of closantel: purity注98.0%.2.2.9 Stock standard solution: Weigh 10 mg standard (accurate to 0.1 mg) (2.2.酌.dissolve with 5 SN/T 1628-2005 acetonitrile to scale in 20 mL volumetric flask. The concentration of soluton is O. 500 mg/mL. The solution should be stored at 1 t -4t and keep sta
15、ble for one year. 2.2. 10 Medium standard solution: Accurately suck 1.00 mL stock standard solution (2.2.酌，dilut with acetonitrile to scale in 50 mL volumetric flask. The concentration of solution is 10.0g/mL. The solution should be stored at 1 t .4 t and keep stable for oneear. 2.2.11 Working stand
16、ard solution: According to the requirement , dlut the medium standard solu tion (2.2.10) with acetonitrile to working standard solution , use immidiately after preparation. 2. 3 Apparatus and equi阳nent.2.3. 1 High-performance chromatograph: equipment with fluorescence detector. 2. 3. 2 Ultrasonic cl
17、eanser. 2. 3. 3 Rotary vacuum evaparator. 2. 3. 4 Nitrogen evaparator. 2.3.5 pH meter. 2. 4 Procedure 2.4. 1 Extraction Weigh ca 5 9 of test臼mple(accurate to 0.01 g) into a 100 mL coni臼Iflask with a cap , add 50 mL acetonitrile(2.2. 1) and 10 9 anhydrous sodium sulfate. Strongly shake the conical fl
18、ask to scatte the sample after臼ppingit, and extract 10 min in ultrasonic cleanser. Then filter the extract through rapid paper into a 150 mL separating funnel. 2. 4. 2 Clean-up Add 40 mL n-hexane (2.2.6) into the separating funnel , cap it tightly, degas after shaking several times lightly. After sh
19、aking for 2 min, let it stand for 10 min. Drain out a part of the acetonitrile laer to a conical flask. Accurately suck 10 mL from it to a test tube, evaporate to drness with nitro gen evaparator at 50 oC. Residues are dissolved with 1. 0 mL acetonitrile in ultrasonic bath. Then the solution is pass
20、ed through O. 45m filter and readto HPLC analsis. 2. 4. 3 Determination 2. 4. 3. 1 HPLC operating conditions a) Column: C，8lumn5m4.6mmx1田mm;Suitable for州=2.5 mobile phase, or the饲uival朋t.6 SN/T 1628-2005 b) Mobile phase: acetonitile-water (75+ 25) + 0.05% triethlamine. The pH value is adjusted to 2.
21、 5 with H3 P04 while measured with a pH meter. c) Flow rate: 1.0 mL/min. d) Fluorescence detector wavelength: EX: 335 nm EM: 510 nm. e) Injection volume: 20L 2. 4. 3. 2 HPLC determination According to the approximate concentration of closantel residues in sample , select the working standard solutio
22、n with similar responses to that of sample solution. The reponses of closantel in the working standard and the sample solution should be within the linear range of the instrument. The working standard solution and the sample solution should be injected alternatively. Under the above HPLC operating c
23、onditions, the retention time of closantel is about 17.6 min. The chromatogram of the standard solution is shown in annex A. 2. 4. 4 Blank test The operation of the blank test is the same as described in determination, but with the omission of sample addition. 2. 4. 5 Calculation and expression of r
24、esults Calculation the content of closantel residue in test sample by HPLC data processor or according to formula (1) X = (A - Ao) C , V As m 飞/aE /，、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . where: X-the residue of closantel in test sample , mg/kg; A一一一thepeak area of closantel of s
25、ample solution , mm勺Ao-the peak area of blank test, mm勺As一一-thepeak area of closantel of standard solution , mm勺C一一-theconcentration of closantel in standard solution，g/mL; V一一-thefinal volume of the sample solution , mL; m一-thecorresponding sample weight in the final solution , g. 3 Limit of determ
26、ination and recover 3. 1 Limit of determination The limit of determination of this method is O. 1 mg/kg. 3. 2 Recovery 3. 2. 1 The recovery in beef sample The recovery range is 86. 2% to 92. 0% , when fortified at the concentration of O. 1 mg/炮，7 SN/T 1628-2005 0.5 mg/kg and 1.0 mg/kg. 3.2.2 The rev
27、ery of cotton臼mpleThe recoverrange is 84.9% to 95. 0% , when fortified at the concentration of O. 1 mg/kg, O. 5 mg/ kg and 1.0 mg/kg. 8 SN/T 1628一2005Annex A (informative annex) Chromatogram of the standard solution -。口创。-u60 40 20 。2属WEHmm 18 16 14 12 10 8 6 4 2 20 0 9 HPLC chromatogram of the closantel standard solution Fig. A.1 mCON-NFHZm 中华人民共和国出入境检验检疫行业标准进出口肉及肉制晶中氯佩础柳肢残留量测定方法高效液相色谱法SN/T 1628-2005 * 中国标准出版社出版发行北京复兴门外三里河北街16号邮政编码:100045网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷晤印张1字数16千字2005年11月第)次印刷开本880X12301/16 2005年11月第一版* 定价10.00元如有印装差错由本社发行中心调换版权专有侵权必究举报电话:(010)68533533书号:155066.2-16472
