1、叶己|中华人民共和国出入境检验检疫行业标准SN/T 1114 - 2002 进出口水果中烯睡醇残留量的检验方法Determination of diniconazole residues in fruits f or import and export 2002-020发布2002斗1-01实施中华人民共和国发布国家质量监督检验检瘦总局SN/T 1114-2002 前言本标准是按照GB/T1. 1-2000(标准化工作导则第1部分:标准的结构和编写规则及SN /T 0001-1995(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
2、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。测定低限是根据国际上对葡萄中烯嗖醇残留量的最高限量和检测方法的灵敏度而制定的。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并归口。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:熊芳、戴华、李拥军、黄志强、张莹。本标准系首次发布的出入境检验检疫行业标准。SN/T1114-m02 1 范围进出口水果中烯睡醇残留量的检验方法本标准规定了进出口水果中烯瞠醇残留量检验的抽样、制样和高效液相色谱测定方法。本标准适用于进出口葡萄中烯瞠醇残留量的检验。2 抽样和制样2.1 检验批以不超过1500件为
3、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 抽样敢量(见表1)襄1批量最低抽样数1-25 1 26-100 5 101-250 10 251-1500 15 2.3 抽样方法按2.2规定的抽样件数随机抽样，逐件开启。每件中至少取500g作为原始样品，原始样品总量不得少于2kg。加封后，标明标记，及时送实验室。2.4 试样制备将所取混合原始样品缩分出1kg，经组织捣碎机捣碎，均分成两份，装入洁净容器内，作为试样。密封并标明标记。2. 5 试样保存将试样于一18C以下避光保存。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
4、定方法3. 1 方法提要试样中残留的烯唾醇用丙嗣提取，经液液分配和固相萃取小柱净化后，用配有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:HPLC级。3.2.2 乙睛:HPLC级。3.2.3 丙酣。1 SN/T 1114-2002 3.2.4 正己烧。3. 2. 5 5%硫酸铀溶液:称取50g硫酸铀用水溶解稀释至1000 mL。3. 2. 6 丙酣正己烧掘合液:1十1，V/V。3. 2. 7 烯瞠醇标准品:纯度注99%。3. 2. 8 烯瞠醇标准溶液:准确称取适量烯嗤醇标准品，用甲醇榕解并配成浓度为1000g/m
5、L的标准储备液。根据需要再配成适当被度的标准工作溶液。3.2.9 活性炭小柱:SUPELCLEANENVI-CARB小柱，125mg , 3 mL或相当者。3. 2. 10 中性氧化铝小柱:SUPELCLEANENVI-ALUMINA-N小柱，125mg , 3 mL或相当者。3.3 仪器和设备3.3.1 高效液相色谱仪，配紫外检测器。3. 3. 2 固相萃取装置，带真空泵。3.3.3 离心机:3000 r/min o 3.3.4 涡旋棍匀器。3.3.5 捣碎机。3.3.6 微量注射器:50L。3. 4 测定步骤3.4.1 提取称取试样约5g(精确到0.01g)于25mL离心管中，加入5mL丙
6、酣，在1昆匀器上提匀1min，于2 500 r/min离心3min，将上清液倒人50mL离心管中，残渣用2X5mL丙酣重复处理两次，合并提取液。3. 4. 2 净化在提取液中加入10mL 5%硫酸锅榕液(3.2.日，再加入7mL正己烧，于棍匀器上混匀1min , 2 500 r/min离心3min后，用尖嘴吸管将上层正己炕相移至另一30mL试管中，用正己皖共提取三次。合并正己烧相，并于50C下空气流中浓缩至近干，用2mL丙酣-正己烧棍合液(3.2.6)溶解残渣。自上而下依次将活性碳小柱(3.2.9)、中性氧化铝小柱(3.2.10)串联接好，安装在固相萃取的真空抽滤装置(3.3.2)上，保持洗脱
7、流速为2mL/min。用8mL丙酣正己烧混合液(3.2.6)活化小柱后，于真空装置下放好收集管，将2mL样品提取液加到小柱上，用3X2mL丙酣-正己皖氓合溶液(3.2.6)洗涤试管并转移至小柱中，再用2X2mL丙酣-正己烧1昆合溶液(3.2.6)淋洗小柱，收集洗脱液。将洗脱液于50C下空气流中吹干，用甲醇1.0 mL定量榕解残渣，过0.45m滤膜后，供高效液相色谱测定。3. 4.3 测定3.4. 3. 1 色谱条件a) 色谱柱:C18柱，250mmX4. 6 mm(内径)，粒径5m，或相当者;b) 色谱柱温度:40C;c) 流动相:乙腊-水(73十27)，过0.45m微孔滤膜zd) 流速:0.
8、5mL/min; e) 检测波长:250nm; f) 进样体积:20L。3. 4. 3. 2 色谱测定根据样液中烯瞠醇的含量情况，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烯唾醇的响应值均应在仪器的检测线性范围内。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，烯唾醇的保留时间约为11.5min。标准品色谱图见附录A中图A.1。3. 4. 4 空白实验除不加试样外，均按上述测定步骤进行。2 3. 5 结果计算和襄述用色谱数据处理机或按下式(1)计算试样中烯瞠醇的含量:x = A X Cs X V 一A. X m 式中zX一一试样中烯瞠醇的含量，mg/kg;A一一样液中烯唾醇
9、的峰面积，mm2;C.一一标准工作液中烯瞠醇的浓度，用/mL;A.-一标准工作液中烯哇醇的峰面积，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。4 谢定低限及回收率4. 1 测定低限本方法对烯瞠醇的测定低限为0.02mg/kg。4.2 回收率在葡萄中烯瞠醇的添加浓度及其回收率的实验数据:在0.02mg/kg时，回收率为87.0%;在0.20mg/kg时，回收率为90.4%;在2.0mg/kg时，回收率为93.0%。SN/T 1114-2002 ( 1 ) 3 SN /T 1114-2002 附录(资料性附录)标准晶色谱固A 面的国-Z箍卸载m
10、Abs 301 Ch1 250nm 罢-2120 10 。-rvmm 20 15 10 烯瞌酶标准晶色谱固固A.14 SN/T 1114-2002 Foreword This standard was drafted in accordance with the requirements of GB/T 1 .1一2000:Directives for of standardization-Part 1: Rules for the structures and drafting of standards and SN/T 0001-1995 General rules for draftin
11、g the standard methods for the determination of pesticide, veterinary drug residues and biotoxins in commodities for expo同 The method of determination of this stan dard was drafted by referring to relevant domestic and foreign literatures through research , modifi cation and verification .Jn additio
12、n , methods of sampling and sample preparation are also specified in this standard. The limit of determination is defined in this standard on the basis of the current international maxi mum limits for diniconazole residues in grapes and the sensitivity of the method. The annex A of this standard is
13、an informative annex. This standard was proposed band is under the charge of Certification and Accreditation administration of the People s Republic of China. This standard was drafted by Hunan Entry-Exit Inspection and Quarantine Bureau of the People s Republic of China. The main drafters of this s
14、tandard are Xiong Fang , Dai Hua , Li Yongjun , Huang Zhiqiang and Zhang Ying. This standard is a professional standard for entry-exit inspection and quarantine promulgated for the first time. Note:This English version ,a translation from the Chinese text ,is solely for guidance. 5 SN/T 1114-2002 De
15、termination of diniconazole residues in fruits for import and expo民1 Scope This standard specifies the methods of sampling , sample preparation and determination of dini conazole residues in fruits for import and export bhigh peormance liquid chromatograph. This standard is applicable to the determi
16、nation of diniconazole in grape for import and export. 2 Sampling and sample preparation 2.1 Inspection lot The quantity of an inspection lot should not be more than 1 500 packages. The characteristics of the cargo within the same inspection lot, such as packing , mark, origin, speci fication and gr
17、ade, should be the same. 2.2 Quantity of sample taken (See Table 1) Table 1 Number of packages in each inspection lot Minimum number of packages to be taken 1 - 25 1 26陶1005 101 - 250 10 251 - 1 500 15 2.3 Sample procedure A number of packages specified in 2.2 are taken at random and opened one by o
18、ne. The sample weight taken as the primary sample from each package should be at least 500 grams. The total weight of all primarsample should not be less than 2 kg , which shall be sealed, labeled and sent to laboratory in time. 2.4 Preparation of test sample The combined prima即sampleis reduced to 1
19、 kg , the po同ionsare blended and divided into two e qual p。同ions.Each portion is placed in a clean container as the test sample, which is then sealed and labled. 2.5 Storage 01 test sample The test samples should be stored below - 18:. 6 SN/T 1114-2002 Note: In the course of sampling and sa町、pleprep
20、aration , precautions must be taken to avoid contamination or any factors which may cause the change the diniconazole residue content. 3 Method of determination 3.1 Principle The diniconazole residues in grapes are extracted by acetone. The extract is cleaned up bliquidliquid extraction and solid ph
21、ase extraction before HPLC determination with UV detector, using ex ternal standard method. 3.2 Reagents and materials U nless otherwise specified , all regents should be anal叽icallypure. Water should be redistilled. 3.2.1 Methanol : HPLC grade. 3.2.2 Acetonitrile: HPLC grade. 3.2.3 Acetone. 3.2.4 n
22、-Hexane. 3.2.5 5% Sodium sulfate (Na2S04) solution: Dissolve 50.0 9 of Na2S04 in 1 000 mL water. 3.2.6 Acetone-n-hexane mixture: 1 + 1, V/V. 3.2.7 Diniconazole: purity99%. 3.2.8 Diniconazole standard solution: Accurately weigh an adequate amount of diniconazole standard , dissolve in methanol and pr
23、epare a solution of 1 000g/mL with methanol as the stan dard stock solution. Dilute the standard stock solution to the required concentration as the standard working solution. 3.2.9 Active carbon cartridge:SUPELCLEAN ENVI-CARB Sep-Pak cartridge , 125 mg ,3 mL or equivalent. 3.2.10 Nuetral alumina ca
24、rtridge: SUPELCLEAN ENVI-ALUMINA-N Sep-Pak cartridge, 125 mg, 3 mL or equivalent. 3.3 Apparatus and equipment 3.3.1 High pe斤。rmanceliquid chromatograph , equipped with the UV detector. 3.3.2 Solid phase extraction with mechanical vacuum pump. 7 SN/丁1114-20023.3.3 Centrifuge:3000 r/min. 3.3.4 Vortex
25、Mixer. 3.3.5 High-speed blender. 3.3.6 Micro-syringe:50L. 3.4 Predure 3.4.1 Extraction Weigh ca 5 9 ofthe test sample(accurate to 0.01 g) into a 25 mL centrifuge tube, add 5 mL of ace tone , blend for 1 min, centrifugalize at 2 500 r/min for 3 min. Transfer the upper clear extract into a 50 mL centr
26、ifuge tube.Repeat the procedure with 5 mL of acetone twice,combine the extracts. 3.4.2 Clean up Add 10 mL of sodium sulphate solution(3.2.5) into the the acetone extracts , extract 3 times with n-hexane,7 mL each time and centrifugalize at 2 500 r/min for 3 min. Combine n-hexane extracts into anothe
27、r 30 mL centrifuge tube and blow nearly dry with air at 50C . Dissolve the residue with 2 mL acetone-n-hexane solution(3.2.6). Linked an active carbon car.tridge(3. 2. 9) and a neutral alumina cartridge (3.2.10) from up to down and set up them to the solid phase extraction (3.3.2). First wash the ca
28、rtridges with 8 mL acetone-n-hexane solution (3. 2. 6) , then transfer the sample residue 2 mL into the cartridges. Wash the test tube with 3 x 2 mL acetone-n-hexane(3.2.6) and add into the cartridges. Wash the cartridges with 2 x 2 mL acetone-n-hexane(3. 2. 6). Collect the eluent and blow dry with
29、air at 50 C . Dilute quantitatively to 1.0 mL with methanol and filter through 0.45m membrane filter. The solution is used for HPLC determination. 3.4.3 Determination 3.4.3.1 HPLC conditions a) HPLC column:C18,250 mm x 4.6 mm(id) ，5m particle size , or equivalent; b) Column temperature:40C; c) Mobil
30、e phase:Acetonitrile-water(73 + 27)，而Iterthrough 0.45m membrane filter; d) Flow rate:0.5 mL/min; e) Detector wavelength: 250 nm; f) Injection volume:20L 3.4.3.2 Determlnation B SNI丁1114-2002According to the approximate concentration of diniconazole in the sample solution, select the stan dard workin
31、g solution with similar peak area to that of the sample solution. The response of dini conazole in the standard working solution and sample solution should be within the linear range of the instrumental detection. The standard working solution should be randomly injected in-between the injections of
32、 the sam ple solution of equal volume. Under the above operation condition, the retention time of dinicona zole is about 11.5 min. For chromatogram of the standard ,see Figure A.1 in annex A. 3.4.4 Blank test The operation of the blank test is the same as that described in the method of determinatio
33、n but without additional of the sample. 3.5 Calculation and expression of the result The operation of the content of diniconazole is carried out by a data processor or according to for mula (1). XZAx c.x V A.x m 、，a咀 ，E、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
34、. where X一一theresidues content of diniconazole in test sample, mg/kg; A-the peak area of diniconazole in the sample solution, mm2; Cs一一-theconcentration of diniconazole in the working standard solution，g/mL; A.-一一thepeak area of diniconazole in the standard working solution, mm飞V-final volume of the
35、 sample solution , mL; m一一massof test sam ple , 9 . Note: The blank value should be substracted from the result of calculation. 4 Limit of determination and recovery 4.1 Limit of determination The limit of determination of this method is 0.02 mg/kg. 4.2 Recovery According to the experimental data ,
36、the fortifying concentrations of diniconazole in grapes and its corresponding recoveries are: 0.02 mgl阔，therecove叩87.0%;0.20 mgl阔，therecove叩90.4%;2.0 mgl阅，therecovery 93.0% . 9 SN/T 1114-2002 Annex A ( i n10rmative) Chromatogram 01 standards 国凹的.时间mAbs 301 Ch1 250nm -DN6ESE-Q SN.OHa且20 。-fv10 mm 20
37、15 10 Figure A. 1-Chromatogram 01 diniconazole standard 10 NOON-叮=同Zm中华人民共和国出入境检验检疫行业标准进出口水果中烯瞌醇残留量的检验方法SN/T 1114-2002 陪中国标准出版社出版北京复兴门外三里河北街16号邮政编码:100045电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷峰开本880X12301/16 印张1字数21千字2002年9月第一版2002年9月第一次印刷印数1-20007巳峰定价10.00网址书号:155066 2-14718 版权专有侵权必究举报电话:(010)68533533
