1、哥也吨盟国境号盒行;佳SN/0152-2014 代替SN0152 1992 71 2，4血?由点V缸VBgq真组ethodfor inspection of 2, 4-D residues in fruit for export 2014-01阳13发布2014-08-01实施A，:俨伪町、。飞户一百十NY 叫阴阳?记:-:J伯母二:/3时;几叫予W-:.!8231正叫中华人提共和国发布国家庭建量监督棱撞撞霞总属SN/01522014 自Ijr=l 本标准按照GB/T1.1一2009给出的规则起草。请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别这些专利的责任。本标准代替SN01
2、52-1992(出口水果中2，4-滴残留量检验方法。本标准与SN0152-1992相比，主要变化如下:一一文本结构不同:SN 0152-1992按GB/T1.1-1987编写，本标准按GB/T1. 1-2009和GB/T 20001.4-2001编写;扩大了检测对象的范围;删除了抽样内容(见1992年版的第2章); 一一增加了样品保存的内容(见第6章); 一一衍生试剂由正丁醇和三氟化棚溶液改为正丁障和浓硫酸;一弗罗里硅土氧化铝层析柱换成弗罗里硅土固相萃取小柱用于样品净化;增加了方法的测定低限和回收率(见第8章)。本标准由国家认证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国湖南出
3、入境检验检疫局。本标准主要起草人:丁利、付善良、张帆、焦艳娜、颜鸿飞、龚强、朱绍华、张莹。本标准所代替标准的历次版本发布情况为:SN 0152-1992。I SN/0152-2014 出口水果中2，4-滴残留量检验方法1 范围本标准规定了苹果、梨、香蕉、菠萝和柑捕中2，4-滴农药残留量的气相色谱测定方法。本标准适用于苹果、梨、香蕉、夜萝和柑桶中2，4-滴农药残留量的测定。2 规范性引用文件下列文件对于本文件的应用是必不可少的。凡是注目期的引用文件，仅注日期的版本适用于本文件。凡是不注日期的引用文件，其最新版本(包括所有的锋改单)适用于本文件。GB/T 6682 分析实验室用水规格和试验方法/
4、3 方法提要试样用磷酸-丙酣溶剂提取，经液液分配净化，以浓硫酸为催化剂，用正丁醇将2，4-滴醋化成2，4-滴丁醋，再经弗罗里硅土固相萃取小柱净化，用气相色谱仪测定，外标法定量。4 试剂和材料除另有规定外，试剂均为分析纯，试验用水应符合GB/T6682三级水指标。4.1 丙翻:液相色谱级。4.2 磷酸:85%。4.3 三氯甲烧。4.4 乙醋。4.5 盐酸:36%38%。4.6 浓硫酸:优级纯，95%98%。4.7 正了醇。4.8 正己烧:液相色谱级。4.9 氢氧化铀。4.10 磷酸氢二铀。4.11 元水硫酸铀:650 oc灼烧4h后，保存于干燥器中。4.12 磷酸水溶液0+1)。4.13 盐酸水
5、溶液o十1)。4.14 磷酸盐溶液:称取2g氢氧化铀(4.9)和44g磷酸氢二铀(4.10)，以水溶解，稀释至1000 mL。4.15 硫酸铀溶液(20g/L):称取2g无水硫酸铀(4.11)溶于100mL水中。4.16 丙酣-正己烧。十的。4.17 2，4-滴标准品:纯度大于等于99.0%，CAS号:94-75而704.18 2，4-滴标准储备液:准确称取0.01000 g 2，4-滴标准品(4.17)于10mL容量瓶中，用约8mL丙酣溶解，用丙酣定容至刻度，得到浓度为1.0mg/mL的标准储备液，于4oc冰箱中保存。SN/T 0152-2014 4.19 2，1-滴标准中间溶液:准确吸取1
6、00L标准储备被(4.18)于100mL容量瓶中，用丙酿定容至刻度，得到浓度为1.0g/mL的标准中间溶液，于4oc冰箱中保存。4.20 2，4滴标准工作液:根据检测需要移取一定体积的标准中间溶液逐级稀释成适当浓度的标准工作溶液。标准工作溶液需现配现用。4.21 弗罗里硅土固相萃取柱:500mg, 3 mL，或相当者。5 仪器和设备5.1 气相色谱仪:配电子捕获检测器(ECD)。5.2 组织捣碎机。5.3 涡旋混匀器。5.4 离心机:6500 r/min。5.5 囡相萃取装置。5.6 全自动浓缩仪。5.7 电子天平:感量为0.1mg ,O.Ol g。5.8 聚四氟乙烯塑料离心管:50mL。5.
7、9 玻璃试管:50mL，具塞。5.10 玻璃刻度试管:5mL，具塞。5.11 旋转蒸发仪。6 试样制备与保存6.1 试样制备从所取全部样品中取有代表性样品500g，将其可食用部分切碎后，用组织捣碎机将样品加工成浆状。?昆匀，装入洁净的盛样容器内，密封并标明标记。在制样的操作过程中，应防止样品受到污染或发生残留物含量的变化。6.2 试样保存将试样于一18oc以下冷冻保存。7 测定步骤7.1 提取称取5g(精确至0.01g)试样于50mL离心管(5.8)中，加入1mL磷酸水榕液(4.12)和10mL丙酿(1.1)，于涡旋泪匀器上快速振荡提取3min，离心3min(6 500 r/min)，将提取液
8、转移到旋蒸瓶中。残渣再用2X10 mL丙酬重复提取两次，合并提取液。在40oC 71-浴旋转浓缩挥干丙酬，残留榕液转入另一离心管(5.8)中，如l入5mL三氯甲烧(4.3)，振蔼提取3mi口，静置分层后，取出三氯甲:境层于-50 mL玻璃试管(5.9)中，残留物再用2X5日lL三氯甲:境(4.3)提取两次，合并三氯甲:皖提取被于现璃试管中。向玻璃试管中加入5mL磷酸盐榕液(4.14)，振荡1mi口，离心3min(2 000 r/min)，将上层水相转入另一玻璃试管(5.的中，三氯甲烧层再用2X5mL磷酸盐溶液(4.14)提取两次，合并水相，用盐酸水榕液(4.13)调pH至12，加入10mL乙酿
9、(4.的，振荡1mi丑，静置分层后，取出乙酿提取液于另一玻璃试SN/0152-2014 管中(5.的，下层水相再用2X 10 mL乙瞠(4.4)重复提取两次，合并乙雕提取液，加入5g无水硫酸铀(4.11)脱水后，转入旋蒸瓶，40oC水浴旋转浓缩至约3mL，转入玻璃刻度试管(5.10)中，用2mL乙醋分两次洗涤旋蒸瓶内壁，洗涤液并入刻度试管中，用全自动浓缩仪拔缩至干。7.2 醋化向上述暖璃刻度试管中加160L正丁薛(4.7)和40L浓硫酸(4.的，加塞混匀后于室温下放置50 min进行丁醋化衍生反应。加入2mL正己烧(4.肘，快速涡旋1min，离心2min(3 000 r/min)。将上层(正己
10、烧层)转移到另一玻璃刻度试管(5.10)中，下层用2X2mL正己烧(4.8)重复提取两次，合并提取液，浓缩至约1mL，待净化。7.3 净化在弗罗里硅土固相萃取柱中加入约1cm厚的无水硫酸铀(4.11)，先用5mL丙酣正己烧(1十9)淋洗，弃去淋洗蔽，然后加入醋化跤，用5mL丙隅正己烧(1十9)洗脱，收集全部洗脱液子玻璃刻度试管(5.10)中，定容至5mL后，供GC-ECD测定。7.4 测定7.4.1 气相色谱条件气相色谱条件如下:a) 色谱柱:日P-5石英毛细管柱，30mXO.32 mm(内径)XO.25m(膜厚)，或相当者;b) 升握程序:初始温度80oC，保持1min，以10oC/mi口的
11、速率升至230oC (不保留)，再以30 oC/min上升至260oC，保持6min; c) 进样口?且度:260oC; d) 检测器温度:300oC; e) 载气:氮气，纯度二三99.999% ，流量1.0mL/min; f) 尾吹气:40mL/min; g) 进样方式:分流进样，分流比为5: 1; h) 进样量:1L。7.4.2 色谱测定根据样液中2，4滴农药的含量情况，选定浓度相近的2，小滴标准工作溶液。标准工作榕液和样液中2，4-滴农药的响应值均应在仪器检测的线性范围内。当样品浓度超出校准曲线线性范围时，将样品稀释至校准曲线线性范围内再测定。对标准工作液和样液等体积交替进样测定，以保留
12、时间定性，以色谱峰面积按外标法定量。在上述气相色谱条件下，2，企滴丁醋化衍生物的参考保留时间为15.37min, 其色谱图参见附录A中图A.1。7.5 空白试验按上述有关步骤进行试剂空白试验。7.6 结果计算用色谱工作站或按式(1)计算试样中2，4-滴农药的含量:一一AsXm . ( 1 ) 3 SN/T 0152-2014 式中:X一一试样中2，4滴农药的残留量，单位为毫克每千克(mg/kg); A 样液中2，4-滴农药的峰面积;c 标准工作液中2，4-滴农药的浓度，单位为微克每毫升(g/mL); V 样液最终定容体积，单位为毫升(mL); As -标准工作液中2，4-滴农药的峰面积;m一一
13、一最终样液所代表的试样量，单位为克(g)。注:计算结果应扣除空白值。8 测定低限和回收率8.1 测定低限本方法的测定低限为0.01mg/峙。8.2 回收率回收率的实验数据:/ / / / / / a) 苹果中2，4町滴添加浓度和自收率数据:添加浓度在O.01 mg/kg 2. 0 mg/同时，回收率为82.8%93.5% ; b) 梨中2，4-滴添加浓度和回收率数据:添如浓度在0.01mg/kg 2. 0 mg/同时，回收率为83.9%98.0% ; c) 香蕉中2，4-滴添加泼皮和回收率数据:添加浓度在0.0.1mg/kg0.05 mg/kg时，回收率为83.1%90.0% ; d) 波萝中
14、2，4-滴添加浓度和回收率数据:添加浓度在0.01mg/kg0.05 mg/kg时，回收率为79.9%95.1% ; e) 柑楠中2，4-滴添加浓度和回收率数据:添加浓度在0.01mg/kg5.0 mg/kg时，因收率为83.8% 97.0%。4 SN/0152-2014 附录(资料性附录)标准溶语气相色谱圈A Hz 280 260 240 220 200 。性的.的叫180 160 140 120 8 盯111116 15 2，4-濡掠准品(0.01阿/mL)丁黯化衍生物色i普图14 13 12 11 10 圈A.l9 5 SN/T 0152-2014 For告word丁hisstandar
15、d was drafted according to the rules specified in GB/T 1.1一2009.Please note that certain contents of this standard may involve patents; the issuance organization of this standard shall not undertake the responsibility of identifying these patents. This standard is the revision of SN 0152-1992 Method
16、 for the determination of 2 ,4-0 residues in fruit for export. Main changes made in this standard are as follows: -The structure of text is different:SN 0152-1992 was drafted according to the rules specified in GB/丁1.1-1987，whilethis standard was drafted according to the rules specified in GB/T 1.1-
17、2009 and GBjT 20001 .4-2001 ; - The application scope is enlarged; - The content relevant to sampling is deletedCChapter 2 in SN 0152-1992); 一Thecontent relevant to the storage of test samples is added CChapter 6); -Replace n西butylalcohol-boron trifluoride solution with n-butI alcohol and sulphuric
18、acid for the derivatization Cesterification) of 2 ,4-0; -Replace florisil column-neutral aluminum oxide column with florisil SPE column for c1 ean up; -Add the limit of determination and recoverof methodCChapter 7). This standard was proposed by and is under the charge of the Certification and Accre
19、ditation Admin istration of the Peoples Republic of China. This standard was drafted bHunan Entry-Exit Inspection and Ouarantine Bureau of the Peoples Re public of China. The main drafters of this standard are Oing Li, Fu Shanliang , Zhang Fan , Jiao Yanna , Yan Hongfei , Gong Oiang , Zhu Shaohua ,
20、Zhang Ying. The releases of all editions substituted by this standard: -SN 0152-1992. 6 SNj0152-2014 1 Scope Method for insp母ctionof 2,4-0 r母sidu母Sin fruit for包xportThis standard specifies the method for determination by gas chromatography spectrometrCGC) of 2,4-0 residues in fruitCapple , pear ,ban
21、ana , pineapple and orange) for export. This standard is applicable for the determination of 2,4-0 residues in fruit Capple , pear , banana , pine apple and orange) for export. 2 Quoted normative documents 丁hefollowing documents are necessary for this standard. For dated reference , only dated editi
22、ons shall apply to this standard.For undated references ,the latest edition of the normative documentCin cluding subsequent amendments) is referred to applies. GBjT 6682 Water for analytical laboratory use一-Specificationand test methods 3 Principle The 2,4-0 residues are extracted from the fruit sam
23、ple with phosphoric acid and acetone solution , followed by liquid-liquid partitioning. Then 2,4-0 in the extract is derived into 2,4-0 butI ester by n butanol and using concentrated sulphuric acid as catalyst. The solution is cleaned up with a florisi I col umn ,and is determinated by gas chromatog
24、raph with ECO.External standard method is used for quan tltatlve付leasure门lent.4 Reagents and materials Unless otherwise specified ,all the reagents used should be analyticallpure，and the water used shall be the grade m specified in GBjT 6682. 4.1 Acetone: HPLC grade. 4.2 Phosphoric acid:85%. 7 SN/01
25、52-2014 4.3 Trichloromethane. 4.4 Ethyl ether. 4.5 Chlorhdric acid:36%-38%. 4.6 Sulphuric acid:95% -98%. 4.7 n-butyl alcohol. 4.8 n-hexane:HPLC grade. 4.9 Sodium hdroxide. 4.10 Oisodium hdrogen phosphate. 4.11 Anhydrous sodium sulfate:lgnite for 4 h at 650 oC ,and keep in a tightly closed container.
26、 4.12 Phosphoric acid-water soluton 口令1). 4.13 Chlorhdrc acid-water solutionC1 +1). 4.14 Phosphate solution: Weigh 2 9 sodium hdroxde(4.9) and 44 9 disodium hydrogen phosphate (4.10) ,dissolves and diluted to 1 000 mLwithiwater. 4.15 Sodium sulfate solution (20 g/L) : Weigh 2 9 anhdrous sodium sulfa
27、te (4.11) , dissolves and diluted to 100 mL with water. 4.16 Acetone-n-hexane solution (1 + 9). 4.17 Standard of 2,4-0:Purity ;?:99.0% ,CAS:94-75-7. 4.18 Stock standard solution of 2.4-0:Accurately weigh 0.010 00 9 2.4-0 (4.17) .dissolve in about 8 mL acetone in volumetric flask individually. and ma
28、ke up to 10 mL with acetone. The concentration of the solution is 1.0 mg/mL. should be stored at 4 oC in refrigerator. 4.19 Standard middle solution of 2.4-0: Suck accuratel100L stock standard solution(4.18) into a 100 mL volumetric flask.dilute with acetone to scale and mix well. The concentration
29、of the solution is 1.0g/mL. should be stored at 4 oC in refrigerator. 4.20 Standard working solution of 2,4-0:According to the requirement. is prepared from the stand ard middle solution bn-hexane and diluted to the required concentration. 8 SN/T 0152-2014 4.21 Florisil SPE column:500 mg ,3 mL, or e
30、quivalent. 5 Apparatus and equipment 5.1 Gas chromatograph spectrometry:equipped with electron capture detector (ECD). 5.2 Tissue triturator. 5.3 Vortex mixer. 5.4 Centrifuge:6500 r/min. 5.5 Solid phase extraction device. 5.6 Automatic concentrated apparatus. 5.7 Analytical balance:0.1 mg ,0.01 g. 5
31、.8 Polytetrafluoroethylene tube:50 mL 5.9 Glass test tube: 50 mL , with glass stopper. 5.10 Glass graduated test tube:5 mL,with glass stopper. 5.11 Rotary vacuum evaporator. 6 Preparation and storage of test samples 6.1 Preparation of test samples The representative sample is reduced to ca 500 g. Th
32、en the edible portions is cut up and homogenized thoroughly in a tissue triturator.Place the homogenized sample in a clean container as the test sam ple , which is sealed and labeled. During the operating process , pollution or change of the residues should be prevented. 6.2 Storage of test samples
33、The test samples shall be stored below -18 oC. 9 SN/0152-2014 7 Procedure 7.1 Extraction Weigh ca 5 9 of the test sample (accurate to 0.01 g) into a 50 mL centrifuge tube (5.8).Add 1 mL phosphoricacid-water solution (4.12) and 10 mL acetone (4.1) , blend for 3 min on a vortex mixer and centrifuge fo
34、r 3 min at a rate of 6 500 r/min. Transfer the extract into a volumetric flask and repeat the extraction for two times more with 10 mL acetone each time.Combine the extract solution to the volumetric flask and eliminate acetone with a rotarevaporator at 40 oC.了ransferthe residue into an other centri
35、fuge tube (5.酌，add5 mL trichloromethane(4.韵，blend for 3 min and let stand to separa ting.Collect trichloromethane laer into a glass test tube (5.9).Repeat this process two more times with 5 mL trichloromethane each time and combine the trichloromethane extract solution into the glass test tube. Add
36、5 mL phosphate solution (4.14) into the glass test tube, and blend for 1 min.After centrifuging for 3 min at 2 000 r/min , transfer upper phase into another glass test tube(5.9) . Repeat the extraction two times more with 5 mL phosphate solution (4.14) each time and combine the extract solution.Ad j
37、usting pH of the extract solution to 1. 2 using chlorhydric acid-water solution (4. 13). Add 10 mL ethI ether (4.4) , blend for 1 min, and let stand to separating. Transfer the extract to another glass test tube (5.9).Repeat the extraction two more times with 10 mL ethI ether each time and combine t
38、he extract. After removing water busing 5 9 anhydrous sodium sulfate (4.11) , transfer the ethyl ether extract to a volumetric flask and concentrate to about 3 mL with a rotarevaporator at 40 oC , Transfer the extract into a glass graduated tube (5.10).Clean the volumetric flask twice with 2 mL ethI
39、 ether in total and combine the cleaning solution to the glass graduated tube.Concentrate the ex tract to dry with an automatic concentrated apparatus. 7.2 Esterification Add 160L n-butI alcohol(4.7) and 40L sulphuric acid (4.6) into the glass graduate tube.Plug the tube with a glass stopper and ble
40、nded on the vortex mixer, then stand for 50 min to complete the de rivatization reaction.Add 2 mL n-hexane (4.8) to the glass graduate tube , blend for 1 min and centri fuge for 2 min at 3 000 r/min.Transfer the upper layer to another glass graduate tube (5.10).Repeat the extraction two times more w
41、ith 2 mL n-hexane(4.8) each time and combine the extract.Concen trate the extract solution to about 1 mL with the automatic concentrated apparatus. The concentrated extract solution is readfor SPE clean up. 7.3 SPE clean up Elute a florisil column (with a laer of anhdrous sodium sulfate about 1 cm t
42、hick placed on top) with 5 mL of acetone-n-hexane (1十9)and discard the eluted solution.Transfer the esterification solution to the column and begin to collect the eluted solution with a glass graduate tube. Elute the column with 5 mL acetone-n-hexane (1 +9).The eluted solution is made up to 5 mL and
43、 is readfor GC-ECD 10 SN/0152-2014 deter1nation. 7.4 Determination 7.4.1 GC operating condition GC operating condition is as following: a) Chromatographic column: Fused silica capillary column , HP-5 , 30 m x 0.32 mm (id) X 0.25m (film thickness) or equivalent; b) Temperature programme: Initial temp
44、erature 80 oC ,hold for 1 min , ramp at 10 oC /min to 230 oC , no hole,then ramp at 30 oC/min to 260 oC ,hold for 6 min; c) Injection port temperature:260 oC; d) Oetector temperature:300 oC; e) Carrier gas: Nitrogen , purity注99.999%，1.0mL/min; f) Make up gas:Nitrogen,40 mL/min; g) Injection mode:Spl
45、it,split ratio:5 : 1; h) Injection volume: 1L 7.4.2 GC determination According to the concentrations of 2，4回0，select the standard working solution with similar peak area to that of sample solution.The responses of 2 ,4-0 standard working solution in the standard working solution and the sample solut
46、ion should be within the linear range of the instrumental detection.Oilute and reanalyze samples with 2 ,4-0 concentration exceeding the range of the calibration curve.Results for such reanalses should fall within the mid崎rangeof the calibration curve. The standard working so lution should be inject
47、ed randomly in between the injections of sample solution of equal volume.Re tention time is applied for qualitative analysis ,and external standard method is emploed for quanti tative analysis.Under the above GC operating conditions ,the retention time of 2，4胃口(as derivative products) is about 15.37
48、 min. For chromatogram of the standard see figure A. 1 in annex A. 7.5 81ank test The operation of the blank test is the same as that described in the method of determination , but with the omission of sample addition. 11 SN/T 0152-2014 7.6 Calculation and expression of result The calculation of res
49、ult is carried out bGC data processor or according to the formula (1) : Where: X=AXCXV As X m X - The residue content of 2，今oin the test sample , mg/kg; A - The peak area of 2,4-0 in sample solution; C - The concentration of 2 , 4-0 in standard working solution,lJg/mL; V - The final volume of the sample solution,mL; As - The peak area of 2,4-0 in standard working solution; m -Mass of test sample , g. Note: The bla
