1、中华人民共和国出入境检验检疫行业标准SN/T 1605-2005 进出口植物性产品中氨草津、氟草隆、劳去津、敌碑、利谷隆残留量检验方法高效液相色谱法Inspection of cyanazin, f1uometuron , atrazine, propanil and Iinuron residues in products of plant origin for import and export-HPLC 2005-08-18发布060608000128 2006-02-01实施中华人民共和国发布国家质量监督检验检瘦总局前言本标准附录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
2、会提出并归口。本标准由中华人民共和国上海出入境检验检疫局负责起草。本标准主要起草人:郭德华、李披、俞秋蓉、韩丽、王敏、王传现、王东辉、魏玉瑛。本标准系首次发布的出入境检验检疫行业标准。SN/T 1605一2005I 1 范围进出口植物性产品中氨草津、氟草隆、劳去津、敌碑、利谷隆残留量检验方法高效液相色谱法SN/T 1605-2005 本标准规定了进出口粮谷中氧草津、氟草隆、劳去津、敌碑、利谷隆残留量的抽样、制样和测定方法。本标准适用于进出口小麦、大麦、大豆、油菜籽和大米中氟草津、氟草隆、劳去津、敌碑、利谷隆残留量的检验。2 抽样和制样2. 1 栓验批以不超过4000袋为一检验批。同一检验批的商
3、品应具有相同的特征，如包装、标记、产地、规格和等级等。2.2 抽样数量按式(1)确定抽样数量。式中zN一一全批袋数pa一一抽样袋数。注:a值取整数，小数部分向前进位为整数。2.3 抽样工具a =.fN . ( 1 ) 2.3. 1 金属单管取样器:不锈钢管，全长55cm(包括手柄)，直径1.5 cm，沟槽长度应超过袋对角线长度的一半。2.3.2 取样铲。2.3.3 分样板。2.3.4 样品筒(袋):可密封。2.3.5 分样布或适用铺垫物。2.4 抽样方法2.4.1 倒包抽样从堆垛的各部位随机抽取2.2规定的应抽样件数的10%(每批一般不少于3袋)，将袋口缝线全部拆开，平置于分样布或其他洁净的铺
4、垫物上，双手紧握袋底两角，提起约成45。倾角，倒拖约1m，使袋内货物全部倒出。查看袋内和袋间品质是否均匀。确认情况正常后，用取样铲随机在各部位抽取样品，立即将样品倒入盛样器内。每袋抽取样品数量应基本一致。2.4.2 袋内抽样按2.2规定的应抽样袋数的90%，在堆垛四周上、中、下各层以曲线形走向随机抽取。将取样器(2.3. 1)管槽朝下，从每袋一角依斜对角方向插入袋内，然后将管槽旋转朝上，抽出取样器，立即将样品倒人盛样器内。每袋抽取样品数量应与2.4.1基本一致。2.4.3 大样缩分集中倒包抽样和袋内抽样所取全部样品，倒于分样布上，用分样板按四分法缩分出样品不少于SN/T 1605-2005 2
5、峙，盛于样品筒内，加封后，标明标记并及时送交实验室。2.5 试样制备将样品按四分法缩分出约1峙，全部磨碎并通过20目筛，混匀，均分成两份试样，装入洁净的容器内，密封，标明标记。2.6 试样保存将试样于50C以下避光保存。3 测定方法3. 1 方法提要试样中的残留物用乙睛提取后过中性氧化铝小柱，浓缩后经固相萃取小柱净化，乙腊-水为流动相，高效液相色谱-质谱/质谱仪测定，用多反应监测(MRM)模式检测，外标法定量。3.2 试剂和材料除另有规定外，试剂均为液相色谱纯，水为重蒸锢水。3.2. 1 乙睛。3.2.2 甲醇。3.2.3 中性氧化铝:100目200目，层析级。3.2.4 氧草津标准品:纯度9
6、8%。3.2.5 氟草隆标准品z纯度98%。3.2.6 秀去津标准品:纯度98%。3.2.7 敌碑标准品z纯度98%。3.2.8 利谷隆标准品:纯度98%。3.2.9 氧草津、氟草隆、芳去津、敌梅、利谷隆标准溶液:分别准确称取适量的氧草津、氟草隆、劳去津、敌碑、利谷隆标准品，用乙脯配成浓度为1mg/mL的标准储备液。3.2. 10 氟草津、氟草隆、秀去津、敌碑、利谷隆标准棍合溶液:准确移取适量上述标准储备液，用乙腊配成浓度为10g/mL的标准混合液。再以乙睛稀释成适用浓度的标准工作溶液。3.3 仪器和设备3.3. 1 高效液相色谱仪，配有四极杆串联质谱仪。3.3.2 离心机:5000 r/mi
7、n。3.3.3 高速均质器。3.3.4 固相萃取装置。3.3.5 筒型漏斗。3.3.6 C18固相萃取小柱:6mL、200mg。3.3.7 氮吹仪。3.3.8 旋转蒸发仪。3.4 测定步骤3.4. 1 提取准确称取已研细样品4.0g于50mL离心管中，加25mL乙腊后，用均质机以10000 r/min的速度均质5min。以2000r/min的速度离心15mino上清液通过装有5g中性氧化铝的筒型漏斗过滤至100 mL蒸发瓶中，滤渣再用20mL乙睛振荡提取5min.重复上述操作，合并上清液于同一蒸发瓶中，于50C水浴减压浓缩至约2.5mL.加入30mL水，混匀。3.4.2 净化在真空固相萃取装置
8、上连接C18固相萃取小柱，依次用6mL甲醇、10mL水活化，将上述样液转移至C18固相萃取小柱上，用10mL 10%的甲醇水溶液淋洗，弃去流出液。分析物用5.0mL甲醇洗脱并SN/T 1605-2005 收集，洗脱液40.C氮气流吹至近干。残留物用1.0 mL乙腊溶解，过0.45m微孔滤膜，滤液供液相色谱-质谱/质谱测定。3.4.3 测定3.4.3. 1 渡相色谱质谱条件a) 色谱柱:ODS-CI8(5m) ,250 mmX 2.1 mm(内径)或相当者;b) 流动相z乙腊-水(60十40);c) 流速:0.25mL/min; d) 柱温z室温;e) 进样量:20L;f) 电离方式:ESI十;
9、g) 毛细管电压:3.53kV; h) 锥体电压:31V; i) 离子源温度:100.C; j) 干燥温度:420.C;k) 雾化气:氯气，纯度99.9%，流量:67L/h; 1) 干燥气z氮气，纯度99.9%，流量:376L/h; m) 碰撞能量:20eV; n) 测定方式:MRM;。)定性离子对和定量离子对见表1。襄1定性离子对和定量离子对除草剂名称定性离子对(m/z)利谷隆249/160 249/182 氟草隆233/72 233/188 秀去津216/174 216/96 敌事事2181162 218/127 佩草津241/214 241/132 3.4.3.2 渡相色谱质谱/质谱测定
10、定量离子对(m/z)249/182 233/188 216/96 218/162 241/132 根据样液中被测氧草津、氟草隆、劳去津、敌碑、利谷隆含量情况，选定峰高相近的标准工作溶液。标准工作榕液和样液中银草津、氟草隆、劳去津、敌碑、利谷隆响应值均应在仪器检测线性范围内。对标准工作溶液和样液等体积参插进样测定。在上述色谱条件下，氧草津、氟草隆、芳去津、敌梅和利谷隆的保留时间分别约为4.03min、4.80min、5.57min、7.34min和8.38min，标准品质谱图参见附录A中图A.1和附录B中图B.1。定性测定，样液如果检出的色谱峰的保留时间与标准溶液中某种除草剂相一致，并且所选择的
11、子离子均出现，而且之间的丰度比也相一致，则可判定试样中含有该种除草剂。3.4.4 空白试验除不加试样外，均按上述测定步骤进行。3 SN/T 1605一20053.4.5 结果计算和表述用色一谱数据处理机或按式(2)计算样品中利谷隆、氟草隆、莞去津、敌碑、氧草津残留含量，计算结果应将空白值扣除。AXcXV x=一一一一一一. .( 2 ) A. Xm 式中zX一一一样品中氟草津、氟草隆、秀去津、敌碑、利谷隆的含量，单位为毫克每千克(mg/kg); C一-氟草津、氟草隆、秀去津、敌碑、利谷隆标准工作液的浓度，单位为微克每毫升(g/mL);A一一样品中氧草津、氟草隆、秀去津、敌碑、和j谷隆的峰面积3
12、A. -_辄草津、氟草隆、秀去津、敌碑、利谷隆标准工作液的峰面积FV-一样液最终定容体秧，单位为毫升(mL);m一一最终样液所代表的试样量，单位为克(g)。4 测定低限、回收率4. 1 测定低限本方法中氟草津、氟草隆、葬去津、敌碑、利谷隆的测定低限均为0.01mg/kg。4.2 回收率在小麦、大麦、大豆、油菜籽、大米中氧草津、氟草隆、秀去津、敌碑、和j谷隆的添加浓度及其回收率实验数据见表2。表2实验数据襄除草剂名称添加浓度!(mg/kg)回收率范围利谷隆0.01 77.3%-88.5% 0.10 83.6%-98.6% 1. 00 88.4%-102.5% 氟草隆0.01 79.8%-89.5
13、% 0.10 75.3%-92.2% 1. 00 86.2%-98.7% 秀去津0.01 79.5%-86.8% O. 10 77.8%-90.7% 1. 00 80.5%-87.4% 敌碑0.01 79.5%-86.9% 0.10 76.8%-90.6% 1. 00 80.5%-95.4% 佩草津0.01 80.6%-89.7% 0.10 83.9%-95.7% 1. 00 80.5%-95.0% 一一一一-一一一一4 附录A(资料性附录)标准晶LC-MS/MS总离子流固100 3 % 5 2 。2.00 4.00 6.00 8.00 10.00 12.00 注:色谱峰号1，2，3，4，5分
14、别为佩草津、氟草隆、劳去津、敌碑和利谷隆e圄A.l佩草津、氟草障、劳去津、敌碑、利谷隆的TIC图SN/T 1605-2005 MRM of 10 Channels ES+ TIC Time 14.00 5 SN/T 1605一20056 附录B资料性附景标准晶色谱圈MRM of 10 Channels ES+ 8. 38 249. 00 160. 00 1 1. 7ge5 %司I 。习J、目目目1111目!11111111目，.l l目I目目.，.M剧而i也注relsES+ WO 8才2249. 00 182. 00 3 1 1. 06e5 %斗I 。斗./-4.03 MRM of 10 Ch
15、annels ES+ 100 241. 00132. 00 I 6.03e5 %斗I, 。司J、MRM of 10 Channels ES+ 4.03 100 ,- 24 1. 00 214.00 I :1. 8ge6 %-=1 1 01J、hMRM of 10 Channels ES+ 4.80 100 233.00 72. 00 司I 4. 85e6 %斗1, 01J、MRM of 10 Channels ES+ 4.80 100 .V 233. 00 188. 00 1 1. 40e5 %斗I, 01J、7. 34 MRM of 10 Channels ES+ lOO 218.00127
16、.00 司17.3ge4 %斗1 01J、L7.34 MRM of 10 Channels.S:+， lOO 1 218.00162.00 习1&OO %斗I 01J飞L5. 57 MRM of 10 Channels ES+ 100 216. 00 96. 00 t 2. 52e6 %-=1 I , 0-1J、5.57 MRM of 10 Channel旧ES+1003 216.00174.00 %斗I 8.68e6 0斗J、1, 111111. i 1II1 i l i i i 1.1 i 11111 i i 11 i ji ii i 1I事I目目川，目芒1I目111I 1 1 1 1目，
17、.I l 尸丁me1. 00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 注:氯草津、氟草隆、秀去津、敌梅和利谷隆的保留时间分别约为4.03min、4.80min、5.57 min、7.34min和8.38 min。固B.1 佩草津、氟草障、费去津、敌穗、利谷隆标准晶的MRM固SN/T 1605-2005 Foreword Annex A and annex B of this standard is an informative an1ex嘻This standard was proposed by and is under the charge of t
18、he Certification and Accreditation Admin istration of the People s Republic of China. This standard was drafted bShanghai Entry-Exit Inspection and Quarantine Bureau of the Peoples Republic of China. The main drafters of this standard are Guo Dehua , Li Bo , Yu Qiurong , Han Li, Wang Min, Wang Chuan
19、xian , Wang Donghui and Wei Yupu. This standard is a professional standard of entry-exit inspection and quarantine promulgated for the first time. Note: this English version, a translation from the Chinese text , is solely for guidance. 7 SN/T 1605-2005 Inspection of cyanazin , fluometuron , atrazin
20、e , propanil and linuron residues in products of plant origin for import and export -HPLC 1 5cope This standard specifies the methods of sampling , sample preparation and determination of canazin， fluometuron, atrazine, propanil , linuron residues in plant origin products. This standard is applicabl
21、e to the determination of cyanazin , fluometuron , atrazine, propanil , linuron residues in wheat, barley , soybean , coleseed and rice for import and export. 2 5ampling and sample preparation 2. 1 Inspection lot The quantity of an .inspection lot should not exceed 4 000 packages. The characteristic
22、s of the cargo within the same inspection lot, such as packing , mark, origin, spec ification and grade, should be the same. 2. 2 Quantity of sample taken Sampling taken is according to the formula (1). a = VN formula: N一一-totalnumber of bags in an inspection lot; a-一-numberof bags to be taken. ( 1
23、) Note: If value a is with decimal , round off the decimal part, which is added as unity to the integral闰rtof a. 2. 3 Sampling tools 2. 3. 1 Metallic sampler: stainless steel; length (including handle): 55 cm; diameter: 1. 5 cm; groove length: longer than half the diagonal length of the bag. 2. 3. 2
24、 Sampling shovel 2. 3. 3 Plate for quartering 8 SN/T 1605-2005 2.3.4 Sample container: Can or bag , which臼nbe sealed. 2.3.5 Cloth (or other suitable material) sheet: For sample dividing (quartering) 2. 4 Sampling procedure 2. 4. 1 Sampling bempting out Draw 10 percent of the number of bags specified
25、 in 2. 2 (not less than three bags) at any part of the pile at random. Unseal and open the bag , and place it on the sampling cloth sheet (or other clean sheet) , Grasp tight two corners of the bag 5 bottom and raise up to an angle of 45 degree , tug back ward for ca 1 m until all ntents of the bag
26、is emptied out. Check whether the quality of goods is uniform within and between the bags. After confirming the goods are in normal condition , scoop up the sample from different parts of the out-poured content at random , and promptly place in a clean sample container. The quantity of the sample dr
27、awn from each bag should be basically the same. 2. 4. 2 Sampling from inside the bags Draw the samples from 90 percent of the number of bags specified in 2.2 (bdeducting the numbet of bags drawn in 2.4.1.1). Along the sine wave of the pile, draw the samples from the bags of the upper, middle and low
28、er pats around the pile at random. Insert the sampler, with its groove facing downward, diagonally into each bag , then turn the sampler by 180 degree , draw out the sampler, and promptly pour the sample into a container. The quantity of the sample drawn from each bag should be basically the same as
29、 in 2. 4. 1. 2. 4. 3 Reduction of gross回mplePour all of samples on a clean sheet , reduce to not less than 2 kg with a plate bquartering. Place in a sample container, seal , label and sent to the laboratory in time. 2. 5 Preparation of test sample Reduce the sample to臼1kg by quartering , grind thoro
30、ughly and let pass through a 20 mesh sieve, mix thoroughly and divide into 2 equal portions. Each portion is placed in a clean container as the test sample, seal and label. 2. 6 Storage of test sample The test samples should be stored below - 50C and kept away from light. 3 Method of determination 3
31、. 1 Principle Residues in sample were extracted with acetonitrile, and pass through a neutral aluminum oxide col umn. After concentrated , the solution is cleaned up by solid-phase extraction column. Determination is made by HPLC-MS/MS with the mobile phase of acetonitrile-water, more reaction monit
32、or (MRM) model , external standard method. 9 SN/T 1605-2005 3. 2 Reagents and materials Unless otherwise specified , all reagents used should be analytically pure. Water is redistilled wa ter. 3.2.1 Acetonitrile: HPLC grade. 3.2.2 Methanol: HPLC grade. 3.2.3 Neutral aluminum oxide column: 100 mesh-2
33、00 mesh , for chromatogra阶y.3.2.4 Canazin standard: Purity98%. 3.2.5 Fluometuron standard: Purity98%. 3.2.6 Atrazine standard: Purity98%. 3.2.7 Propanil standard: Purity98%. 3.2.8 Linuron standard: Purity98%. 3.2.9 Cyanazin , fluometuron, atrazine, propanil , linuron standard solution: Accurately we
34、igh an adequate amount of standard separately , dissolve in acetonitrile and prepare a solution of 1 mg/mL in concentration as the standard stock solution. 3.2. 10 Cyanazin , fluometuron , atrazine, propanil , linuron standard mixture solution: Prepare a standard mixture solution of 10g/mL in concen
35、tration by diluting the above stock solutions with acetonitrile, then dilute to suitable concentration with acetonitrile as standard working solution. 3. 3 Apparatus and equipment 3.3. 1 High performance liquid chromatograph , equipped with quadrupole MS/MS tandem spec trometer. 3.3.2 Centrifuge:500
36、0 r/min. 3.3.3 High-speed homogenizer. 3.3.4 Solid-phase extraction apparatus. 3.3.5 Cylinder filler. 3.3.6 Solid-phase extraction column: C8 6cc, 200 mg , or equivalent. 10 SN/T 1605-2005 3. 3. 7 N2 evaporator 3. 3. 8 Rotary evaporator 3. 4 Procedure 3. 4. 1 Extraction Weigh 4. 0 9 of the test samp
37、le grinded thoroughly into a 50 mL centrifuge tube. Add 25 mL acetoni trile and homogenize for 5 min at 10000 r/min. Then centrifuge mixture at 2 000 r/min for 15 min. The supernatant is filtrated into a 100 mL evaporator by passing through a cylinder filler containing 5 9 of neutral aluminum oxide.
38、 Add 20 mL acetonitrile to remanet in 50 mL centrifuge tube and re peat above procedure. Combine the supernatant to evaporator and concentrate it to the volume of 2.5 mL on a rotary evaporator at 500C , adding 30 mL water and mixed solution. 3. 4. 2 Cleanup Place solid-phase extraction column C,s on
39、to solid-phase extraction apparatus and is conditioned with 6 mL methanol and 10 mL water. Pass the above solution through C,s column ,wash with 10 mL 10% methanol-water and discard the effluents. Wash and collect the effluents with 5.0 mL methanol , and evaporate the effluents to dryness under a ge
40、ntle stream of nitrogen at 40 C. The residue is resolved with 1.0 mL acetonitrile, filtered with 0.45m membrane and ready for HPLC-MS/MS determina ton. 3.4.3 Determination 3. 4. 3. 1 HPLC-MS/MS operating conditions a) Column : ODS-C,s (5m) ,250 mm x 2.1 mm (i. d. ) or equivalent; b) Mobile phase:Ace
41、tonitrile-water (60+40); c) Flow rate:O. 25 mL/min; d) Column temperature:at room temperature; e) Injection volume: 20L; f) Electron ionization: ESI + ; g) Capillary voltage:3. 53 kV; h) Cone voltage:31 V; i Source temperature: 1000C ; j) Desolvation temperature:420C ; 的Conegas:N2 ,Purity99. 9% ,67
42、L/h; 1) Desolvation gas:N2 ,Purity99. 9% ,376 L/h; m) Collision energ:20 eV; n) Detection mode:MRM; 0) Qualitative ions and quantitative ions see table 1. 11 SN/T 1605-2005 Table 1 Qualitative ions and quantitative ions Herbicides Qualitative ions (m/z) Quantitatve ions (m/z) Cyanazin 249/160 249/18
43、2 249/182 Fluometuron 233/72 233/188 233/188 Atrazine 216/174 216/96 216/96 propanil 218/162 218/162 218/127 linuron 241/214 241/132 241/132 3. 4. 3. 2 HPLC-MS/MS determination According to the estimated approximate concentraton of herbcdes n the sample solution. select standard workng soluton of sm
44、lar peak area to that of sample soluton. The responses of herb cdes n the standard workng soluton and the sample solution should be in the linear range of the n strumental detection. The standard working solution should be injected randomly in-between the n jectons of the sample solution of equal vo
45、lume. Under the above chromatographic condition. the re tenton time of cyanazin. fluometuron. atrazine. propanl. linuron s4. 03 min. 4.80 min. 5.57 min. 7.34 min and 8.38 min respectively. For the chromatogram of standard. see fig. A. 1 in annex A and fg. B. 1 n annex B. Confirmaton: If there s a pe
46、ak appeared at the same retenton tme for both of the sample solution and standard workng soluton. and all selected ons appeared and the ratio of abundance s accord ng. confirm the herbcde beng in the test sample. 3. 4. 4 Blank test The operation of the blank test is the same as that described in the
47、 method of determnaton but with the omission of sample addton. 3. 4. 5 Calculation and expression of the result The calculaton of result s carried out by HPLC data processor or according to the formula (2). The blank value should be subtracted from the above result of calculation. 12 where: AxcxV x=
48、 ( 2 ) A. x m X一-theresidue content of cyanazin , fluometuron, atrazine, propanil , linuron in the test sam ple.mg/kg; C一-一theconcentratioin of cyanazin. fluometuron. atrazine. propanil. linuron in the standard workng soluton，g/ml; A一-thepeak area of cyanazin. fluometuron. atrazine, propanl. linuron in the sample solu-SN/T 1605-2005 tion; A.一一-thepeak area of canazin， fluometuron , atrazine, propanil , linuron in the standard work ing solution; V一一一thefinal volume of sample solution,mL; m一一-thecorresponding mass of the test sample in the final sample solution , g. 4 Limi
