1、中华人民共和国出入境检验检疫行业标准SN/T 1624-2005 进出口蔬菜和水果中畸霉股、畸菌肢、睛菌略及畸菌醋残留量检验方法Inspection of pyrimethanil ,mepanipyrim, myclobutanil and azoxystrobin residues in vegetable and fruit for import and export 2005-08-18发布060608000109 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本标准的附录A为资料性附录。.品.r._ 目。自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
2、人民共和国河北出入境检验检疫局起草。本标准主要起草人z郭春海、陈瑞春、王凤池、马振栋、贾海涛。本标准系首次发布的出入境检验检疫行业标准。SN/T 1624-2005 I 1 范围进出口蔬菜和水果中畸霉肢、暗菌胶、腊菌略及嘻菌自旨残留量检验方法SN/T 1624-2005 本标准规定了蔬菜和水果中略毒腊、嘈菌胶、睛菌瞠、暗菌醋残留量检验的抽样、制样与气相色谱和气相色谱-质谱测定方法。本标准适用于苹果、草莓、黄瓜、茄子中唔霉胶、嘈菌胶、腊菌略、嘈菌醋残留量的检验。2 抽样和制样2. 1 检验批以不超过1500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
3、抽样数量抽样数量见表10表1抽样数量批量/件最低抽样数/件125 1 26100 5 101250 10 2511 500 15 2.3 抽样方法按2.2规定的抽样件数随机抽取，逐件开启，每件至少取500g作为原始样品，原始样品总量不得少于2闻，装入清洁密封的样品筒内，加封后标明标记，及时送实验室。2.4 试样制备从混合原始样品中缩分出1kg，取可食部分，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 试样保存将试样于一180C以下冷冻保存。注:在抽样和制样的操作过程中，必须防止样品受到污染或发生残留物含量的变化。3 测定方法3. 1 方法提要用丙酣提取样品中
4、残留的嘀霉腊、暗菌腊、腊菌瞠、暗菌醋，提取液中加入氯化铀溶液，用乙酸乙醋萃取，有机相蒸干，残渣溶解后过ENVI-Carb柱+NHz柱，洗脱液蒸干定容，用气相色谱仪或气相色谱质谱联用仪检测，外标法定量。3.2 试剂和材料除另有规定外，试剂均为分析纯，水为蒸锢水或去离子水。3.2.1 丙酣z优级纯。1 SN/T 1624一20053.2.2 乙酸乙酶z重蒸锢。3.2.3 乙睛z液相色谱级。3.2.4 甲苯。3.2.5 乙睛甲苯(3十1)。3.2.6 氯化铀。3.2.7 氯化铀溶液(200g/L):称取200g氯化锅用蒸馆水溶解并定容至1000 mLo 3.2.8 元水硫酸铀:于650C灼烧4h，冷
5、却后储于密闭容器中备用。3.2.9 正己烧。3.2. 10 丙酣-正己烧0+1)。3. 2. 11 ENVI-Carb柱，500mg, 5 mL. 3.2.12 NH2柱，500mg,3 mL。3. 2. 13 ENVI-Carb柱十NH2柱组合柱:ENVI-Carb柱上部加2cm无水硫酸纳，NH2柱接在ENVICarb柱的下端，用前用15mL乙睛-甲苯(3.2.5)淋洗，备用。3.2.14 嘈霉胶、嘈菌胶、睛菌略、嘈菌醋标准品:纯度注99%。3.2. 15 暗霉腊、暗菌腊、腊菌瞠、喃菌醋标准储备液z准确称取适量的嘈霉腊、暗菌脏、睛菌略、嘈菌醋标准品，用丙酣分别配制成100mg/mL标准储备液
6、，50C以下保存。3.2. 16 嘈霉腊、暗菌腊、腊菌唾、暗菌醋标准工作液:根据被测物含量不同，移取一定量的储备液(3.2.1日制成适当浓度的混合标准工作溶液，50C以下保存。3.3 仪器和设备3.3. 1 气相色谱仪，配氮磷检测器。3.3.2 气相色谱质谱联用仪。3.3.3 平板摇床。3.3.4 璇涡混合器。3.3.5 固相萃取装置。3.3.6 旋转蒸发器。3.3.7 氮吹仪。3.4 测定步骤3.4. 1 提取称取试样约5g(精确到0.01g)于100mL锥形瓶中，加25mL丙酣(3.2.1)，在平板摇床上振摇30 mino过滤入分液漏斗中，用20mL丙酣(3.2.1)分两次洗涤锥形瓶及滤渣
7、，合并滤液于分液漏斗中，于上述分液漏斗中加入25mL氯化锅溶液(3.2.6)和30mL乙酸乙醋(3.2.2)，振摇2min，静置分层，收集有机相。下层水相再用20mL乙酸乙醋(3.2.2)萃取一次，合并有机相，过无水硫酸铀柱至心形瓶中。于旋转蒸发器450C水浴蒸干。3.4.2 净化用4mL乙睛-甲苯(3.2.5)溶解残渣，将榕液转入ENVI-Carb柱NH2柱(3.2.13)，再用4mL乙腊甲苯(3.2. 5)洗心形瓶，待柱中洛液至硫酸纳上部液面时加入柱中，用30mL乙睛-甲苯(3.2.5)洗脱组合柱(3.2.13)，接收全部洗脱液于心形瓶中。于450C水浴上旋转蒸发至干。用3mL丙酣-正己烧
8、(3.2.10)洗心形瓶2次，转入离心管中，在氮吹仪上450C氮气吹干，最后用丙酣-正己烧(3.2.10)定容至1. 0 mL。供气相色谱或气相色谱-质谱分析。3.4.3 测定3.4.3.1 气相色谱条件2 a) 色谱性:石英毛细管色谱柱DB-lMS 30 mXO. 25 mm(内径)X 0.25m(膜厚)或相当者;lO C !min _. 30C /min b) 色谱柱升温程序:200oC(l min)一一:.250.C一一二.285C(10 min); SN/T 1624-2005 c) 进样口温度:2500C ; d) 检测器(NPD)温度:2500C;e) 气体:载气为氮气，纯度99.
9、999%，柱流速3.0mL/min;氢气，纯度99.999%，流速4.0mL/min; 空气，流速90mL/min;尾吹气流速20mL/min; f) 进样方式:不分流;g) 进样量:2L。3.4.3.2 气相色谱-质谱条件3.4.3.2. 1 色谱条件a) 色谱柱:DB-5MS，30mXO. 25 mm(内径)XO. 25m(膜厚)或相当者;30 C /min _ 10C/ b) 色谱柱升温程序:2100C(2min)一一一一280C一一一一:.2900C (6 min); d 进样口温度:250C; d) 载气:氮气，纯度99.999%; e) 载气流速:恒流模式1mL/min; f) 进
10、样方式:不分流;g) 进样量:2L。3.4.3.2.2 质谱条件a) 接口温度:2800C;b) 离子源:电子轰击源(El); c) 电离电压:70eV; d) 离子源温度:2300C ; e) 检测方式:SIM;f) 选择离子及相对丰度:见表2。表2选择离子及相对辛度被测组分略霉胶略哥选择离子(m/z)118 184 198 199 181 208 相对丰度/(%)3 4 100 47 2 5 被测组分脯菌瞠嗜选择离子(m/z)245 150 179 288 344 372 相对丰度1(%)14 53 100 13 100 14 a 为定量离子。3.4.3.3 气相色谱和气相色谱-质谱测定菌
11、胶222 223 100 51 菌醋388 403 28 13 对样液及标准工作被等体积参差进样测定。实际应用的标准工作液和待测样液中，嘈霉胶、暗菌胶、睛菌唾、暗菌醋的响应值均应在仪器的线性范围之内。在上述气相色谱条件下，暗霉胶、喃菌胶、睛菌瞠、暗菌醋的保留时间分别为2.8min、4.8min、5.2min和11.7 mi凡在上述气相色谱-质谱条件下，暗霉腊、喃菌胶、睛菌瞠、暗菌醋的保留时间分别为3.1min、4.5min、4.8min和10.8mino标准品气相色谱图和气相色谱-质谱图参见附录A。3.4.3.4 气相色谱-质谱确证试验进行样品测定时，如果检出的质量色谱峰保留时间与标准样品一致
12、，并且在扣除背景后的样品谱图中，各定性离子的相对丰度比与浓度接近的标准溶液谱图一致，则可判断样品中存在对应的被测物。3.4.3.5 空白试验除不加样品外，按上述相同条件和步骤进行。3 SN/T 1624-2005 3.5 结果计算和表述用色谱数据处理仪或按式(1)计算，计算结果需扣除空白值。式中zX=A.c.V 一A, .m X一一式样中暗霉腊、嘈菌胶、睛菌嗖、暗菌醋含量，单位为毫克每千克(mg/kg); A一-样液中嘈霉胶、暗菌胶、睛菌瞠、暗菌醋的峰面积，单位为平方毫米(mmZ); C一一标准溶液中嘈霉腊、嘈菌胶、脯菌瞠、暗菌醋的浓度，单位为微克每毫升(g/mL); 4 方法的测定低限、回收
13、率4. 1 测定低限. ( 1 ) 气相色谱法和气相色谱-质谱法的测定低限均为:嘈霉胶为0.2mg/kg;暗菌胶为0.2mg/kg;脯菌瞠为0.05mg/kg;嘈菌醋为0.03mg/kg o 4.2 回收率4.2. 1 苹果中四种农药的添加浓度及回收率的数据在0.20mg/kg、0.50mg/kg、5.00mg/kg时，嘈霉胶回收率为85.7%93.8%; 在0.20mg/kg、0.26mg/kg、2.00mg/kg时，唔菌肢回收率为87.2%95. 7%; 在0.05mg/kg、0.18mg/kg、0.50mg/同时，睛菌瞠回收率为85.7%93. 8%; 在0.03mg/kg、0.30mg
14、/kg、0.56mg/kg时，暗菌醋回收率为92.8%96. 8%。4.2.2 草莓申四种农药的添加浓度及回收率的数据在0.20mg/kg、0.50mg/kg、10.00mg/kg时，嘈霉腊回收率为84.3%102. 4%; 在0.20mg/kg、0.26mg/kg、10.00mg/kg时，嘈菌胶回收率为87.1%107.2%; 在0.05mg/kg、0.18mg/kg、1.00 mg/同时，睛菌瞠回收率为91.4%103. 5%; 在0.03mg/kg、0.56mg/kg、5.00mg/kg时，暗菌醋回收率为93.2%102. 5%。4.2.3 黄瓜中四种农药的添加浓度及回收率的数据在0.2
15、0mg/kg、0.50mg/kg、5.00mg/kg时，暗霉胶回收率为89.0%95. 0%; 在0.20mg/kg、0.26mg/kg、2.00mg/kg时，暗菌肢回收率为89.5%93. 8%; 在0.05mg/kg、0.18mg/kg、0.50mg/kg时，腊菌瞠回收率为85.3%94. 4%; 在0.03mg/kg、0.30mg/kg、0.56mg/kg时，暗菌醋回收率为86.5%102. 2%。4.2.4 茄子中四种农药的添加浓度及回收率的数据4 在0.20mg/kg、0.50mg/kg、1.00 mg/kg时，嘈霉肢回收率为82.3%106. 5%; 在0.20mg/kg、0.26
16、mg/kg、5.00mg/kg时，略菌股回收率为82.5%88. 6%; 在0.05mg/kg、0.18mg/kg、1.00 mg/kg时，脯菌略回收率为86.9%99. 4%; 在0.03mg/kg、0.56mg/kg、2.00mg/kg时，暗菌醋回收率为91.8%108. 2%。SN/T 1624-2005 附录A(资料性附录)标准物的气相色i普及气相色谱-质谱图mV 60 40 2 20 3 ._(Il一LI。5 。1 1-嗜霉胶$2 暗菌胶;3一脯菌瞠34 暗菌醋。固A.1标准混合物的气相色谱固Abundance 2 50000 40000 ! UAU nu nu qo 20000 4
17、 3 10000 5000 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 1嗜霉胶;2一瞻菌胶;3一腊菌睡z4 啼菌醋。圄A.2标准混合物的气相色谱-质谱圄(TIC)5 SN/T 1624-2005 6 Abund剧lce900000 700000 500000 300000 51 100000 60 m/z-7000 5000 3000 1500 77 Abundance 260000 220000 180000 140000 100000 60000 20000 198 143 184 118 100 140 180 220 26
18、0 300 340 圈.3晴霉膀标准物的质谱图222 80 120 160 210 230 图.4晦菌股标准物的质谱固179 150 82 55 .且.守160 100 140 180 220 260 280 图.5睛菌睡标准物的质谱圈Abundance 80000 65000 45000 30000 m/z-Abundance 34000 I 30000 26000 22000 14000 10000 6000 Abudance 80000 70000 60000 50000 40000 30000 15000 181 m/z一180 75 80 118 115 185 SN/T 1624-
19、2005 344 388 172 329 372 180 260 340 400 图A.6暗菌醋标准物的质谱固198 184 125 135 145 155 165 175 185 195 205 圄A.7晴霉肢标准物选择离子质谱圈222 208 190 195 200 205 210 215 220 225 230 圄A.8晴菌脏标准物选择离子质谱固7 SN/T 1624-2005 Abundance 7000 179 6000 4000 3000 2000 1000 245 288 。150 160 170 180 190 200 210 220 230 240 250 260 270 2
20、80 290 圄A.9脯菌睡标准物选择离子质i曾圈Abundance 10000 , 344 9000 8000 7000 6000 5000 4000 388 3000 2000 372 403 1000 。340 350 360 370 380 390 400 410 圈A.10晴菌醋标准物选择离子质谱图8 SN/T 1624-2005 Foreword Annex A is an informative annex. This standard was proposed byand is under the charge of State Authentication and Super
21、vised Com mittee of the People 5 Republic of china. This standard was drafted bHebei Entr-Exit Inspection and Ouarantine Bureau of the Peoples Re public of China. The main drafters of this standard are Guo chunhai ,Chen ruichun. Ma zhendong , Wang fengchi and Jia haitao. This standard is an Entry-Ex
22、it Inspection and Ouarantine professional standard published for the first time. 9 SN/T 1624-2005 Inspection of pyrimethanil , mepanipyrim , myclobutanil and azoxystrobin residues in vegetable and fruit for import and export 1 Scope This standard specifies the method of sampling , sample preparation
23、 and determination by gas chro matography and gas chromatograph-Mass spectrum of Pyrimethanil , Mepanipyrim , Myclobutanil and Azoxystrobin residues in vegetable and fruit. This standard is applicable for the detemination of Pyrimethanil , Mepanipyrim, Myclobutanil and Azoxstrobin residues in apple,
24、 strawberry, cucumber and eggplant. 2 Sampling and sample preparation 2. 1 Inspection lot Each inspection lot should not exceed 1 500 packages. The characteristics of the臼rgowithin the same inspection lot,such as packing ,mark,origin,specifi cation and grade,should be the same. 2. 2 Quantity of samp
25、le taken see Table 10 Table 1 Quantit。fsample Number of packages in each inspection lot Minimum number of packages to be taken 1.25 1 26.100 5 101-250 10 251-1 500 15 2.3 Sampling procedure A number of package specified in 2. 2 are taken at random and opened one by one. The sample weight taken as th
26、e primary sample from each package should be at least 500 g. The total sample weight taken as the primary sample should be at least 2炮，placedin a clean sample can , sealed , la beled and send to laboratory in time. 10 SN/T 1624-2005 2. 4 Preparation of te5t sample The conmbined primary sample is red
27、uced to 1闹，theedible portion is blended , and divided into two equal portion. Each portion is placed in a clear container as the test sample, which is sealed and labled. 2. 5 Storage of test sample The test sample should be stored at -18t. Note:ln the course of sampling and sample preparation ,preca
28、ution must be taken avoid the contami nation or any factors which may臼usethe charge of residue content. 3 Method of determination 3.1 Principle After the residues of pyrimethanil ,Mepanipyrim ,Myclobutanil and Azoxystrobin are extracted byac etone from the sample , adding the sodium chloride solutio
29、n into the extracted solution , and extract with ethyl acetate. After the organic phase is vaporized to dryness , disslove the draff then through the column of ENVI-Carb+ amido. The combined solution is vaporized to dryness and exactly diluted. Finally the solution is analyzed by gas chromatograph o
30、r GC-MS , quantitation with external standard method. 3. 2 Reagents and materials Unless otherwise specified , all reagents are analtically pure water is distilled water. 3.2. 1 Acetone: Excellent Pure. 3.2.2 Ethyl acetate: Redistilled. 3.2.3 Acetonitrile: UV-grade. 3.2.4 Toluene. 3.2.5 Acetontrile-
31、toluene(3 + 1). 3.2.6 Sodium chloride. 3.2.7 Sodium chloride solution(200 g/L): Weight 200 9 Sodium chloride ,dissolve and exactly dilu ted to 1000 mL. 3.2.8 Anhydrous sodium sulfate:lgnite at 6500C for 4 h,and keep in a tightly closed container after cooling. 3.2.9 n-hexane. 11 SN/T 1624一20053. 2.
32、10 Acetone-N-hexaneC 1 + 1). 3. 2. 11 Column of ENVI-Carb , 500 mg , 5 mL. 3. 2. 12 Column of N陀，500mg ,3 mL. 3. 2. 13 Column of ENVI-Carb + NH2 : Anhydrous sodium sulfate is putted into Column of ENVI-Carb to 2 cm , connect column of NH2 under Column of ENVI-Carb. Rinse the column of ENVI-Carb + NH
33、2 with 15 mL Acetontrile-Toluene(3. 2. 5). 3.2.14 Pyrimethanil，Mepanipyrim，Myolobutanil.Azoxstrobin standard:Purity99%. 3.2. 15 Pyrimethanil , Mepanipyrim , Myclobutanil , Azoxystrobin standard stock solution: Accurately weigh an adequate amount of four standard ,dissolve in acetone to make up four
34、standard stock solu tions of 100g/mL. The solution should be stored below 50C. 3.2. 16 Pyrimethanil , Mepaniprim. Myclobutanil.Azoxstrobin standard working solution: Accord ing to the content of analyzed matter. make up mixture standard working solution by adding adequate each of the stock solution
35、and dilutng with acetone. The solution should be stored below 50C. 3.3 Apparatus and equipment 3. 3. 1 Gas chromatograph equipped with NPD. 3.3.2 Gas chromatograph equipped with mass spectrograph(GC-MS). 3. 3. 3 Orbital shaker. 3. 3. 4 Minishaker. 3.3.5 Solid phase extraction equipment. 3. 3. 6 Rota
36、ry evaporator. 3. 3. 7 Nitrogen evaporator. 3. 4 Procedure 3. 4. 1 Extraction Weight ca 5 g(accurate to O. 01 g) of the test sample into 100 mL conical flask.adding 25 mL acetone (3.2. 1) .shake for 30 min on a orbital shaker.filtering with funnel ,transfer the filtrate into separator funnel. Rinse
37、the conical flask with 20 mL acetoneC 3. 2. 1) in twice portions ang filter. combine the 12 SN/T 1624-2005 filtrates to the separetor funnel. Add 25 mL sodium chloride solution(3. 2. 6) and 30 mL ethyl acetate (3.2.幻.shake vigorously 2 min. let stand for separating comperately. collect the organic p
38、hase. Wa ter phase was extracted once with 20 mL ethyl acetate(3. 2. 2) .combine organic phase and pass the organic phase through the cloumn of anhydrous sodium sulfate and in a heart-shape bottle. Evaporate the solution to dryness with a rotary evaporator under reduced at 45t water-bath. 3. 4. 2 Cl
39、ean up Oisslove the residue in 4 mL acetontrile-toluene(3. 2. 5). transfer the solution into column of ENVI Carb+ NH2 Rinse the heart-shape bottle with 4 mL acetontrile-toluene(3. 2. 5) .pouring the solution into the cloumn (3. 2. 13) when the solution of cloumn reach the top of anhydrous sodium sul
40、fate. Then elute the cloumn(3. 2.13) with 30 mL acetontrile-tolueneC3. 2.日.andcollet all the elution in a heart-shape bottle. Evapor to dryness with a rotary evaporatol under reduced at 45t water-bath. Rinse the heart-shape bottle twice with 3 mL acetone- N-hexane(3. 2. 10). transfer the solution in
41、to tube.and evaporate to dryness under nitrogen flow at 450C. Make up to 1. 0 mL with acetone- N-hex ane(3. 2.10) promptly for GC or GC-MS determination. 3. 4. 3 Oetermination 3. 4. 3. 1 GC operating conditions a) Capilary column: Fused silica. 08-1 MS .30 m X O. 25 mm( id) X O. 25m (film thickness)
42、 ; b) 10 C /min _O 30 C /min Column oven temperature procedure: 2000C (1 min)一一一-一-250UC一-一一一:.2850C ( 10 min); c) Injection temperature: 2500C ; d) Oetector(NPD) temperature: 250t; e) Gas: Carrier gas Nitrogen. purity 99. 999%. flowrate 3. 0 mL/min; Hydrogen gas. purity 99.999%. flowrate 4. 0 mL/mi
43、n; Air. flowrate 3. 0 mL/min; Makeup gas. flowrate 20 mL/min; f) Injecting formate:Splitless; g) Volume of injection: 2L 3. 4. 3. 2 GC-MS operating conditions 3. 4. 3. 2. 1 GC operating conditions a) Capilary column: Fused silica. D8-5 MS.30 m X 0.25 mm( id) X 0.25m (film thickness); 30 C /min _o 10
44、 C /min b) Column oven temperature procedure:210oC (2 min)-一-一一280UC-一一一-吃90C(6 min); c) Injection temperature: 2500C ; d) Carrier gas:Helium.purity 99. 999 %; e) Carrier gas flowrate: Constant mode 1 mL/min; f) Injection mode:Splitless; g) Injection volume: 2L. 3. 4. 3. 2. 2 MS operating conditions
45、 13 SN/T 1624一2005a) Interface temperature:280C ; b) lon Source: Electron Impact lon Source (EI) ; c) Electron Energy: 70 eV; d) Source temperature: 230oC; e) Detection mode: SIM; f) Sclected ions (m/z) and relative intensity (%): see Table 2. Table 2 Sclected ions and relative intensity anal叭e的rime
46、thanilMepanipyrim SelectedCmjz) 118 1倒198 199 181 208 2228 223 Relative intensityj 3 4 100 47 2 4 100 51 C%) anal叭eMyclobutanil Azoxystrobin SelectedCmjz) 245 150 179 288 344 372 388 403 Relative intensity j 14 53 100 13 100 14 28 13 C%) a IS quantltave ion 3. 4. 3. 3 GC and GC-MS determination The
47、standard woking solution should be randomly injected in-between the injections of the sample solution of equal volume. The respond of Pyrimethanil ,Mepanipyrim , Myclobutanil and Azoxystrobin residues in the standard working solution and the sample solution should be within the linear range of the i
48、nstrument detection. Under the above GC operating condition , the retention time of Pyrimetha nil ,Mepanipyrim,Myclobutanil and Azoxystrobin residues separatly is ca 2.8 min, 4. 8 min,5. 2 min and 11. 7 min. Under the above GC-MS operating condition , the retention time of Primethanil， Mepa nipyrim,
49、Myclobutanil and Azoxystrobin residues separatly is ca 3. 1 min ,4. 5 min ,4. 8 min and 10.8 min. For the chromatogram of the standard , see annex A. 3.4.3.4 GC-MS confirmed examination If the retention times of sample chromatogram peaks are consistent with the standards , and after subtracted background noise ,the relative intensity ratios of each qualitative ions are also consistent with simi
