1、:才己中华人民共和国出入境检验检疫行业标准SN /T 1116-2002 进出口饲料中克伦特罗、沙丁肢醇残留量的检验方法液相色谱法Determination of c1enbuterol and salbutamol residues in animal feed for import and export 一-唱Liquidchromatographic method 2002- 05-20发布2002 -11-01实施中华人民共和国国家质量监督检验检瘦总局发布SN/T 1116-2002 前本标准是按照GB/T1. 1-2000标准化工作导则第1部分z标准的结构和编写规则及SN/T 000
2、1-1995出口商品中农药、兽药残留量及生物毒素检验方法标准编写的基本规定的要求进行编写的。其中测定方法是参考了国内外有关文献，经研究、改进和验证后制定的。本标准同时制定了抽样和制样方法。测定低限是根据液相色谱测定饲料中克伦特罗、沙丁股醇残留量方法的灵敏度而制定的。本标准的附录A和附录B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。本标准由中华人民共和国湖南出入境检验检疫局负责起草。本标准主要起草人z戴华、袁智能、黄志强、陈新焕。本标准系首次发布的出入境检验检疫行业标准。SN/T 1116-2002 进出口饲料中克伦特罗、沙丁肢醇残留量的检验方法液相色谱法1 范围本标准规定了进出
3、口饲料中克伦特罗、沙丁脑醇残留量检验的抽样、制样和液相色谱测定方法。本标准适用于进出口全价饲料和预混饲料中克伦特罗、沙丁胶醇残留量的检验。2 抽样和制样2. 1 检验批以不超过2000件为一检验批。同一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(见表1)2.3 抽样工具2.3.1 取样勺。2.3.2 分样板。2. 3. 3 分样布。批量500以下501-1 000 1 001-2000 2. 3. 4 盛样器:筒或袋，可密封。2.4 抽样方法最低抽样数15 25 30 按2.2规定的应抽样袋数从堆垛的各部位随机抽取样袋，逐件开启。从每袋中用取样勺抽取代表性
4、样品约500g。将抽取样品立即倒入盛样器内。每袋所取样品的量应基本一致，每批所抽取的样品总量应不少于4峙。将所取样品全部倒于分样布上，用分样板按四分法缩分样品至不少于2kg。倒人盛样器内，密封并标明标记，及时送实验室。2.5 试样制备2. 5. 1 制样工具a) 粉碎机。b) 筛子:2mm圆孔筛、20目筛。c) 分样板。d) 盛样器z具塞广口瓶。2.5.2 制样方法将取回样品全部粉碎使通过2mm圆孔筛;t昆匀，用四分法缩分至约500g。继续粉碎使全部通过1 SN /T 1116-2002 20目筛。据匀，用四分法均分成两份，盛于盛样器内作为试样。密封井标明标记。2.6 试样保存将试样于-5C以
5、下避光保存。注:在抽样和制样的操作过程中，必须防止样品受到污染或发生残留物含量的变化。3 测定方法3.1 方法提要试样中残留的克伦特罗、沙丁肢醇经含甲醇的盐酸溶液超声提取，液-掖萃取后，用固相萃取小柱净化，净化液中的克伦特罗、沙丁股醇用配有二极管阵列检测器或紫外检测器的高效液相色谱仪测定，外标法定量。3.2 试剂和材料除特殊规定外，所有试剂均为分析纯，水为重蒸锢水。3. 2. 1 甲醇。3.2.2 乙睛:HPLC级。3.2.3 正己烧。3.2.4 85%磷酸。3.2.5 0.10 mol/L盐酸榕液:取8.33mL浓盐酸溶液用水稀释至1000 mL。3. 2. 6 o. 030 mol/L盐酸
6、溶液:取300mL o. 1 mol/L盐酸溶液用水稀释至1000 mL。3.2. 7 0.10 mol/L盐酸-甲醇溶液z盐酸+甲醇(9十1)。3. 2. 8 2. 0 mol/L氢氧化饷溶液z称取40.0g氢氧化铀用水溶解并稀释至500mL。3.2.9 乙酸乙醋+正丁醇溶液(6+4)，用水饱和。3. 2. 10 o. 007 5 mol/L磷酸二氢饵溶液:pH值3.0。溶解1.02 g磷酸二氢梆于800mL水中，用磷酸(3.2.4)调pH值至3.0，然后用水定容至1000 mL。3.2.11 4% (V /V)氧化甲醇榕液:取4.0mL氨水(密度0.88g/mL)用甲醇稀释至100mL。3
7、.2.12 阳离子交换小柱:SUPELCLEANLC-SCX Sep-Pak小柱，500mg , 3 mL或相当者。3.2.13 盐酸克伦特罗标准品z纯度二三99%。3.2.14 沙丁胶醇标准品:纯度二三99%。3. 2. 15 标准储备液(1000 mg/L):称取0.0570 g盐酸克伦特罗(相当于0.0500 g克伦特罗)于50 mL容量瓶中，用甲醇榕解并定容至刻度;称取0.0500g沙丁胶醇于50mL容量瓶中，用甲醇溶解并定容至刻度，作为标准储备液。根据需要再用甲醇将标准储备液稀释成适当浓度的混合标准工作液。3.3 仪器和设备3.3.1 高效掖相色谱仪，配二极管阵列检测器或紫外检测器。
8、3. 3. 2 超声波水路。J. 3. 3 Sep-Pak真空抽滤装置，带真空泵。3. 3.4 离心机:4000 r/mn。3. 3. 5 混匀器。3. 3. 6 pH 计。3. 4 测定步骤3. 4. 1 提取准确称取5.00g试样于50mL离心管中，加入20mL盐酸-甲醇溶液(3.2.7)，于棍匀器上棍匀后，置超声波水浴中超声30min(期间每10min取出混匀一次)，超声提取后冷却至室温，于2500r/min离心5min，将上清液过滤到50mL容量瓶中，残渣用2X10mL盐酸-甲醇溶液(3.2.7)混匀提取两次，合并提取液，用盐酸，甲醇溶液(3.2.7)洗涤滤纸并定容至刻度。2 SN /
9、T 1116-2002 3.4.2 净化定量取25.0mL提取液于50mL离心管中，加入5mL正己烧，于混匀器上棍匀1min. 1 500 r/min离心5min后，用尖嘴吸管将正己皖相去掉，共处理二次。再用2.0mol/L NaOH溶液(3.2.8)调节提取液pH值至12.用3X5mL乙酸乙醋，正丁醇溶液(3.2.9)提取样液，每次混匀2min. 于2500 r/min离心5min后，用尖嘴吸管将上层有机相合并取到另一离心管中，在(70I5)C下，用氯气流吹至近干，用1mL盐酸-甲醇搭液(3.2.7)溶解残渣。将两根SCX小柱(3.2.12)串联接好安装在真空抽滤装置(3.3.3)上，保持洗
10、脱流速为1mL/min. 依次用5mL甲醇、5mL水和5mL O. 030 mol/L盐酸榕液(3.2. 6)处理小柱F然后将1mL样品提取液加到小柱上;再用1mL盐酸-甲醇榕液(3.2. 7)洗涤试管并一起转移至小柱中;依次用5mL水、5mL 甲醇淋洗小柱。在溶剂流过固相萃取柱后，保持真空抽气状态至少保持5min。于真空装置下放好刻度收集管，用5mL 4%氨化甲醇溶液(3.2.11)洗脱，并收集洗脱液。将洗脱液于(70土5)C下，用氮气流吹至近干，用甲醇溶解残渣并定容至2.0mL.过0.45m滤膜后，供液相色谱测定。3.4.3 测定3- 4. 3- 1 色谱条件a) 色谱柱:CN柱.250m
11、mX4. 6 mm(内径).粒径10m.或相当者;b) 流动相z乙脯+O. 007 5 mol/L磷酸二氢饵榕液(85+15).混匀后用磷酸调节pH值至3.O.过0.45m微孔滤膜;c) 流速:1. 0 mL/min; d) 检测波长:215nm; e) 进样量:20L。3.4. 3- 2 色谱测定根据样液中克伦特罗、沙丁胶醇的含量情况，选定峰面积相近的标准工作溶液。标准工作溶液和样液中的克伦特罗、沙丁肢醇的响应值均应在仪器的检测线性范围内。对标准工作榕液和样液等体积参插进样测定。在上述色谱条件下，克伦特罗、沙丁胶醇的保留时间分别约为13.5min、17.4mino标准品色谱图见附录A中图A.
12、1。标准品紫外吸收光谱图见附录B中图B.1和图B.20 3.4.4 空白实验除不加试样外，均按上述测定步骤进行。3.5 结果计算和囊述用色谱数据处理机或按下式。)计算试样中克伦特罗或沙丁胶醇的含量:V一-m Q一-A A一X . ( 1 ) 式中zX一一试样中克伦特罗或沙丁胶醇的含量.mg/kg;A一一样液色谱图中克伦特罗或沙丁胶醇的峰面积.mm2;C.一一标准工作液中克伦特罗或沙丁胶醇的浓度，用/mL;A. -标准工作液色谱图中克伦特罗或沙丁胶醇的峰面积.mm2;V一一样液最终定容体积.mL;m 一一-最终样液所代表的试样量.g。注:计算结果须扣除空白值。3 SN /T 1116-2002
13、4 测定低限、回收率4.1 测定低限本方法克伦特罗、沙丁胶醇的测定低限分别为0.10mg/kg、0.36mg/kg。4.2 回收率4.2.1 饲料中克伦特罗添加浓度及其回收率的实验数据:在0.10mg/同时，回收率为89.4%;在1.00 mg/kg时，回收率为93.6%; 在8.00mg/kg时，回收率为96.3%。4.2.2 饲料中沙丁胶醇添加浓度及其回收率的实验数据z在0.36mg/kg时，回收率为75.1%; 在3.60mg/kg时，回收率为74.9%; 在28.8mg/kg时，回收率为83.6%。4 SN/T 1116-2002 附景(资料性附录)标准晶色调固A gn.hH槛锺HA树
14、2-nH阻四撑血事MMCh1 215nm mAbs 40 30 20 10 型iJ.-l-. 。5 20 mm 15 克伦特罗、沙丁脏醇标准晶色谱固10 5 。圄A.1SN/T 1116-2002 6 mAbs 22.7 mAbs 31.。211 附录B(资料性附景)标准晶紫外光谱圄220 240 260 280 300 320 340 nm 圄B.1克伦特罗紫外吸收光谱固226 303 334 220 240 260 280 300 320 340 nm 固B.2沙丁肢醇紫外吸收光谱圈SN/T 1116-2002 Foreword This standard was drafted in a
15、ccordance with the requirement of GB/T 1.1-2000 Directives for of standardization-Part 1: Rules for the structures and drafting of standards and SN/丁0001-1995General rules for drafting the standard methods for the determination of pesticide , veterinary drug residues and biotoxins in commodities for
16、 export. The method of determination of this standard was drafted by referring to relevant domestic and foreign literatures through research , modification and verification .In addition , method of sampling and sample preparation are also specified in this standard. The limit of determination is def
17、ined in this standard on the basis of the sensitivity of the method by liquid chromatography. Annex A and annex B of this standard are informative annexes. This standard was proposed by and is under the charge of Certification and Accreditation of the Peoples Republic of China. This standard was dra
18、fted by Hunan Entr-Exit Inspection and Quarantine Bureau of the People s Republic of China. The main drafters of this standard are Dai Hua , Yuan Zhineng , Huang Zhiqiang and Chen Xinhuan. This standard is a professional standard for entry - exit inspection and quarantine promulgated for the first t
19、ime. Note: This English version ,a translation from the Chinese text , is solely for guidance. 7 SN/T 1116-2002 Determination of clenbuterol and salbutamol residues in animal feed for import and expo同-Liquid chromatograpic method 1 Scope This standard specifies the methods of sampling , sample prepa
20、ration and determination of clen buterol and salbutamol residues in animal feed for import and expo同byliquid chromatography. This standard is applicable to the determination of clenbuterol and salbutamol residues in pre mixed feed and fodder feed for import and expo同.2 Sampling and sample preparatio
21、n 2. 1 Inspection lot Each inspection lot should not exceed 2 000 packages. The characteristics ofthe cargo within the same inspection lot,such as packing , mark, origin ,speci fication and grade, should be the same. 2.2 Quantity of sample taken (see Table 1) Table 1 Number of packages in an inspect
22、ion lot Minimum number of packages to be taken below 500 15 501 - 1 000 25 1 001 - 2000 30 2.3 Sampling tools 2.3.1 Sampling ladle. 2.3.2 Plate for quartering. 2.3.3 Sheet:For sample dividing (quartering). 2.3.4 Sample container:Can or bag ,which can be sealed. 8 SN/T 1116-2002 2.4 Sampling procedur
23、e Draw the number of bags specified in 2.2 at any pa民Sof the pile at random , unseal and open the bag one by one. From each bag , ca 500 9 of representative sample shall be taken. Promptly place the samples in a sample container. The quantity of the sample drawn from each bag should be ba sically th
24、e same ,and the total weight of the sample drawn form each lot should be not less than 4 kg. Pour all the sample onto a clean sheet and reduce to not less than 2 kg by quartering with a plate. Place in a sample container, seal , label and send to the laboratory in time. 2. 5 Preparation of test samp
25、le 2.5.1 Tools for sample preparation a) Pulverizer. b) Sieve: 2 mm round - hole sieve , 20 mesh sieve. c) Plate for quartering. d) Sample container: Wide - mouth bottle, with ground stopper. 2.5.2 Procedure Pulverize all the sample taken back to let pass through a 2 mm round - hole sieve , mix and
26、reduce by quartering to ca 500 g. Pulverize again to let all pass thorough a 20 - mesh sieve. Mix thorough Iy and divide into two equal portions, place in clean sample containers as the test samples , seal and label. 2.6 Storage of test sample The test samples should be stored below - 5C and kept aw
27、ay from light. Note : In the course of sampling and sample preparation , precautions must be taken to avoid contamination or any factors which may cause the change of residue content. 3 Method of determination 3.1 Principle The clenbuterol and salbutamol residues are extracted from the test sample b
28、y hydrochloric acid solution containing methanol , and the extract is clean - up by liquid - liquid extraction and solid phase extraction before determination by HPLC with PAD or UV detector, using external standard method. 3.2 Reagents and materials Unless otherwise specified , all reagents should
29、be analytical pure , water is redistilled water. 3.2.1 Methanol. 3.2.2 Acetonitrile: HPLC grade. 9 SNIT 1116-2002 3.2.3 n - Hexane. 3.2.4 85% Phosphoric acid. 3.2.5 0.10 mol/L Hydrochlorc acd solution: Dilute 8.33 mL hdrochloric acid (HCI) to 1 000 mL with water. 3.2.6 0.030 mol/L Hydrochloric acid
30、solution:Dilute 300 mL 0.1 mol/L HCI to 1 000 mL with wa ter. 3.2. 7 Hydrochloric acid - methanol solution: Dilute 100 mL methanol to 1 000 mL with 0.10 mol/L HCI (3.2.5). 3.2.8 2.0 mol/L Sodium hdroxide (NaOH) solution: Disslove 40.0 9 of NaOH in 500 mL water. 3.2.9 Ethyl - acetate + n - butanol so
31、lution (6 + 4) ,Saturated with water. 3.2.10 Potassium dihydrogen phosphate (KH2P04) solution:0.007 5 mol/L at pH value 3.0, Dis solve potassium dihydrogen phosphate in 800 mL water, adjust the pH value to 3.0 with concen trated phosphoric acid (3.2.4) and dil ute to 1 000 m L with water. 3.2.11 4%
32、Ammoniacal methanol:Dilute 4 mL ammonia solution (sp.gr.0.88) to 100 mL with methanol. 3.2.12 Anion - exchange cartridge:SUPELCLEAN LC - SCX Sep - pak cartridge ,500 mg ,3 mL or equivalent. 3.2.13 Clenbuterol hydrochloride:purity;!: 99%. 3.2.14 Salbutamol:purity;!: 99%. 3.2.15 Standard solution (1 0
33、00 mg/L) : Weigh accurately about 0.057 0 9 clenbuterol hydrochlo ride (equivalent to 0.050 0 9 of clenbuterol) standard into a 50 mL volumetric flask. Dissolve and make up to mark with methanol. Weigh accurately about 0.050 0 9 salbutamol standard into a 50 mL volumetric flask. Dissolve and make up
34、 to mark with methanol as the standard stock solution. Dilute the standard stock solution with methanol to the required concentration as the mix stan dards working solution. 3.3 Apparatus and equipment 3.3.1 HPLC equipped with PAD (photodiode array detector) or UV detector. 3.3.2 Ultrasonic bath. 10
35、 SN/T 1116-2002 3.3.3 Sep - Pak vacuum manifold with mechanical vacuum pump. 3.3.4 Centrifuge:4000 r/min. 3.3.5 Mixer. 3.3.6 pH Meter. 3.4 procedure 3.4.1 Extraction Weigh accurately 5.00 9 sample into 50 mL centrifuge tube, add 20 mL hydrochloric acid -methanol solution (3.2.7) .Sonicate for 30 min
36、utes in an ultrasonic bath (shaking at least every 10 min) , let cool to room temperature. Decant solution through filter paper into 50 mL volumetric flask. Repeat extraction of solid with 20 x 10 mL Hdrochloric acid - methanol solution (3.2. 7) shake vigorously, centrifuge and filter supernatant th
37、rough filter paper in the same volumetric flask. Wash the tube and filter cake with small amount of hydrochloric acid - methanol solution (3.2.7) ,and combine the filterates and make up to 50 mL. 3.4.2 Cleanup Pipet 25.0 mL of the extract into 50 mL centrifuge tube.Add 5 mL n - hexane into the centr
38、ifuge tube , mix on a mixer for 1 min , centrifugalize at 1 500 r/min for 5 min , draw out the n - hexane lay er with a capillartipped pipet and discard , repeat the step two times. The pH value was adjusted to 12 by 2.0 mol/L Sodium hydroxide solution (3.2.8).A volume of 3 x 5 mL of ethylacetate -
39、n butanol solution (3.2.9) was added to the extract and the solution was mixed on a mixer for 2 min.The sample was centrifuged (2 500 r/min , 5 min) and the organic phase was collected b capillarytipped pipet. The organic phase was evaporated to near dryness under a stream of nitro gen at (70:!: 5)
40、OC. Dissolve the residue in 1 mL hdrochloric acid - methanol solution (3.2.7). Set up the vacuum manifold and mechanical pump (3.3.3) .Condition two SCX cartridges (in se ries) (3.2.12) by washing with 5 mL methanol ,5 mL water and then 5 mL 0.030 mol/L HCI (3.2. 6) ,then transfer the sample extract
41、 1 mL into the cartridges. Wash the test tube with 1 mL of hy drochloric and make up to 2.0 mL with methanol , and filter through 0.45m membrane filter. The solution is acid - methanol solution (3.2. 7) and add to the cartridge. Wash the cartridges with 5 mL water and then 5 mL methanol. Drain off e
42、xcessive liquid in the cartridges by continuous suc tion for at least 5 minutes. Elute with 5 mL 4% ammoniacal methanol (3.2.11) and collect the eluant in a glass tube with stopper, evaporate the eluate to flear dryness under nitrogen at (70 :!: 5) OC . Dissolve the residues used for HPLC determinat
43、ion. 11 SN/T 1116-2002 3.4.3 Determination 3.4.3. 1 HPLC operating condition a) Column:CN column (25 cmx4.6 mm i.d.，10m pa同iclesize) or equivalent; b) Mobile phase:Acetonitrile + 0.007 5 mol/L K2P04 solution (85 + 1日，thepH value was adjusted to 3.0 by phosphoric acid (3.2.4) , filter through 0.45m m
44、embrane filter; c) Flow rate:1.0 mL/min; d) Detector wavelength: 215 nm; e) Injection volume:20L 3.4.3.2 HPLC determination According to the approximate concentration of clenbuterol and salbutamol in the sample solution , select the standard working solution with similar peak area to that of the sam
45、ple solution. The re sponses of clenbuterol and salbutamol in the mix standard working solution and sample solution should be within the linear range of the instrumental detection.丁hemix standard working solution should be randomlinjected in - between the injections of the sample solution of equal v
46、olume. Under the above operating condition , the retention time of clenbuterol is about 13.5 min and salbutamol about 17 .4 min. For chromatogram of the standard , see Figure A. 1 in annex A. For ul traviolet spectrum of clenbuterol and salbutamol , see Figure B. 1 and Figure B. 2 in annex B. 3.4.4
47、Blank test The operation of the blank test is the same as that describe in the method of determination but without addition of sample. 3.5 Calculation and expression of the result The calculation of the residue content of clenbuterol or saJbutamoJ in the test sample is carried out by HPLC data proce
48、ssor or according to the formula (1): Where: A x Cs x V ( 1 ) - As x m x-一一theresidue content of clenbuterol or salbutamol in the test sample , mg/kg; A-the peak area of clenbuterol or salbutamol in the sample solution , mm飞cs-the concentration of clenbuterol or salbutamol in the standard working so
49、lution，g/ mL; As-一一thepeak area of clenbuterol or salbutamol in the standard working solution mm飞m-the corresponding mass of the sample in the final sample solution g. Note: The blank value should be subtracted from the result of calculation. 4 Limit of determination and recovery 4.1 Limit of determination The limit of determination of clenbuterol is 0.10 mg/kg and salbutamol is 0.36 mg/kg. 12 SN/T 1116-2002 4.2 R部。very4.2.1 According to the experiment data ,
