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1、中华人民共和国出入境检验检疫行业标准SN/T 4655-2016 出口食品中草甘麟及其代谢物残留量测定方法液相色谱-质谱/质谱法Determination of glyphosate and its metablize residues in foodstuffs for export-HPLC-MS/MS method 2016-08-23发布2017-03-01实施中华人民共和国发布国家质量监督检验检疫总局SN/T 4655-2016 目IJ1=1 本标准按照GB/T1.1-2009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。本标准由国家认
2、证认可监督管理委员会提出并归口。本标准起草单位:中华人民共和国厦门出入境检验检疫局检验检疫技术中心、宁波出入境检验检疫局、福建出入境检验检疫局。本标准主要起草人:徐敦明、陈树兵、吴敏、杜凤君、余于成、刘正才、张结、张志刚。1 范围出口食品中草甘瞬及其代谢物残留量测定方法液相色谱-质谱/质谱法SNjT 4655-2016 斗标汗u见过了山口食品rjl草山脚及其代词j物(氨叩基磷酸)残罔忱的液相也语-质语/质语测liJ方法。本标准适用于茶叶、小麦、玉米、稻谷、甘臣、大豆、柑楠、苹果、桃、葡萄、香蕉、西瓜、大麦茶、棉籽油中草甘瞬及其代谢物残(氨甲基磷酸)残留量的检测和确证。2 规范性引用文件下列文件
3、对于本文件的应用是必不可少的。凡是注日期的引用文件，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(包括所有的修改单)适用于本文件。GBjT 6682 分析实验室用水规格和试验方法3 方法提要试样用水提取，经透析袋、RP柱及石墨化碳黑吸附剂净化，用液相色i普-质谱/质谱仪测定，外标法定量。4 试剂材料除非另有说明，所用试开IJ均为分析纯，水为GBjT6682规定的一级水。4.1 甲醇，色谱纯。4.2 乙睛，色谱纯。4.3 乙酸锻.色谱纯。4.4 乙酸镀溶液(5rnrnol/L,pH:10 11):准确称取3.8:)5g(精确至0.0001 g)乙酸错，用水定容至50 rnL，配
4、制成1rnol/L乙股钱济波:再取其1115rnL，JIj水;4:容主1000 rnL，氨水调节pH全1O11。4.5 标准物质:平甘麟(glyphosate，GLY,CAS:1071-83-6)，纯度关08.0%;每甲基磷酸(arninornethyl phosphonic acid,AMPA,CAS:1066-51-川，纯度关98.0%。4.6 透析袋:直径22rnrn，压平宽度(半周长)31rnrn，截留分子量3500 Dao 4.7 标准品溶液的自己制:准确称取GLY和AMPA标准品各10rng，用水定容至10rnL，配置成浓度为1 rng/rnL的标准储备溶液。4.8 标准储诸备的配
5、制:眼取标J准任品、珞溶液，用水稀释分别配制成浓度为1川O问g/凡/4.9 标准工作溶液的配制:根据需要，11临|恼臼用时吸取一定量的标准储备f液夜用水洛j液夜稀释成适当浓度的1混昆合标j准佳工作溶掖。4.10 透析袋处理:用剪刀剪至10crn20 crn小段，用水煮沸10rnin，二次水洗净保存至乙醇中川吏用前后均用二次水清洗净。4.11 OnGuard 1 I RP柱(1.0 cc柱):使用前5mL甲醇、5mL水活化.静置半小时;使用后甲醇清洗SN/T 4655-2016 保存。4.12 石墨化碳黑吸附剂(GCB):拍m60m，ProElul填料，或相当者。4.13 微孔滤膜:0.22nl
6、，有机系，聚四氟乙烯(PTFE)膜o5 仪器与设备5.1 液相色谱质谱/质谱仪:配有电喷雾离子掘(ESI)。5.2 电子天平:感量分别为0.01g,O.OOO 1 g。5.3 组织捣碎机。5.4 粉碎机。5.5 均质器:15000r/mi口。5.6 离心机:i000 min以上。5.7 高速离心机:15000r/min以上。5.8 超声仪。5.9 离心管:i0mLo 5.10 试管:50mL 5.11 注射器。5.12 涡璇提匀器o5.13 氮吹仪。6 试样制备与保存6.1 试样制备6.1.1 液体样品液体样品棍匀备用。6.1.2 果蔬类取代表性样品约500g，将其切碎后，用组织捣碎机将样品加
7、工成浆状，也匀，装入洁净容器，密封，标明标记。6.1.3 粮谷类和茶叶类取代表性样品约500g，用粉碎机粉碎，过20曰筛，由匀，装入洁净容器，密封，标明标记。6.2 试样保存将试样于5飞以下避光保存。注在抽样及制样的操作过程中弓应防止样品受到污染或发生残留物含量的变化。7 分析步骤7.1 提取液体样品、粮谷类、及果蔬样品称取i式样1g(精确到0.01g)，于50mL离心管(5.9)中.加入6mL 2 SN/T 4655-2016 水，于高速(15000 r/min)均质器(5.盯上均质1日山，以15000 r/min离心(5.7)5min，收集水相，用水定容至10mLc 茶叶类样品称取试样2g
8、(精确到0.01g)，于50mL离心管(5.9)中，加入10mL水，于高速(15 000 min)均质器(5.5)上均质3r丑1口，将离心管以15000 r/min离心(5.7)5min，收集上层液.用水定容至10mL。7.2 净化取样品提取液10mL加入经活化了的透析袋(4.10)中，并将透析袋置于己加入10mL水的;)0mL 试管(5.10)中，超声(5.8)1ho取出透析袋，从玻璃管中取出5mL残液，用5mL注射器(5.11)注入己活化的RP柱(4.11)，弃去RP柱前2mL流出液，用5mL离心管收集后3mL流出液;1m入20mg GCB(4.12)，旋窝震荡2min后于15000 r/
9、min离心5min，取2mL上清液至洁净的玻璃管中60oc下氯吹(5.13)至下句残渣用0.5mL的;J(j容液溶解，过聚四氟乙怖(PTFE)膜，供j夜十日色前-民讲l质ff仪CLC-MS/MS)分析。7.3 测定7.3.1 液相色谱-质谱/质谱条件液相色语质谱/质i普条件如下:a)色谱柱:NH2P-502D柱，长150mm，内径2.0mm，粒径5m或相当者;b)流动相:A:5mmol/L乙酸接水梅液(氨7150%士2%20%50%士25%10%20%士3%运10%士50%在仪器最佳工作条件下，对标准工作溶液进样o用标准工作曲线按外标法定量，样品j容液中被测物的响应值均应在仪器测定的线性范围内
10、。根据试样中被测样液的含量情况，选取响应值相近的标准工作液进行色卅分析。标准工作液和样液中待测物的响应值均应在仪器线性响应范围内，如果待测物含量超过线性范围，则重新取样分析，用水稀释到线性也固内后分析。在ij主色i前条件下GLY和AMPA的参考保留时间约为1.68mi口和1.20min，标准溶液的多反应监测(MRM)色谱图参见附录巳。7.4 空白试验除不加试样外，均按上述操作步骤进行o8 结果计算和表达结果用色前数据处型机或按;r:(1)计并:f(中甲:廿腾和纸甲某磷酸的残自量:式中:x=x V X 10 X 1 000 711 X 1 000 X 试样中草甘瞬或氨甲基磷酸残留含量-单位为毫克
11、每千克(mg/kg);c 标准工作洛液中草甘腾或氨甲基磷酸的浓度，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);m 试样称取质量，单位为克(g)。注.计算结果应扣除空白值。9 测定低限和回收率9.1 测定低限本方法对甲:计麟和主l甲某磷酸残臼量的测定低限均为50g/kgo9.2 回收率不同添加浓度范围内回收率的实验数据，见表304.(1)SN/T 4655-2016 表3本方法添加浓度及回收率范围(n=6)添加回收率范围添加回收率范围基质浓度基质浓度mg/kg 单U腾氮甲基瞬酸mg/kg 市un弃交(甲基腾酸。0:576.4%88.3%79.2%88.5%。.0:57
12、7.3%84.9%82.4%90.2%茶叶。.183.2%90.6%85.7%96.8%柑情。.189.1%99.2%8:5.9%91.7%I 82.9%川.0%S4.5%川.:，%1 川.4%川.:，%!J1.:,%川.:，%。.0:578.9%89.2%79.9%90.1%。.0:583.6%93.2%84.8%94.2%。.186.2%93.5%89.9%99.1纠。.190.3%97.2%91.2纠97./1%小麦苹果I 82.6%89.5%86.1川95.7%0.5 91.8%97.6%89.8%96.6%5 94.9%104.1%90.6%剧.2%1 9:U%99.7%91.0%9
13、U%。.0;SO.S%96.2%自:U%9，).2%0.0,)7S.S%SU%79.:%90.2%玉米。.189.3%94.2%89.4%97.2%桃0.1 88.3%95.8%89.9%97.0%95.1%10J.;%89.3%9斗s%86.9纠99.0%90.3%103./1%。.0;76.:，%S!U%SO.2%归.S%。.0，)80.2%川.2%76.6%S:U%稻谷。.183.9%95.1%87.4%94.9%葡萄。.189.5%l01.7%8:5.3%92.7%90.6%99.3%97.2%102.3%8:1.3%97.8%90.2%96.7%。.0;78.3%的.4%77.8%川
14、.3%。.0，)7S.3%S!J.2%80.;%92.7%西瓜。.186.9%97.3%86.9%9斗2%香蕉。.187.0%93.5%8/1.6纠92.6%I 97.3%105.4%89.4%99.2%I 84.9%97.6%81.0%89.5%0.05 79.5%89.9川82.7%92.7%。.0582.5%92.8川83.2%92.8川0.1 80.8%91.0%87.1%93.0%。181.3%90.1%81.9%90.1%大主止U:!1主I 89.4%93.7%86.3川90.4%I 84.7%94.7川86.3%92.9川2 8;.9%9U%85.1%91.4%2 84.2%97
15、.8%86.:,%97.8%。.0;77.2%S!J.8%80.!J%川.S%。.0，)77.2%92.!J%79.2%川.1%大麦89.0%97.9%87.3%94.6 川十南非子79.8%川.4%78.4川87.4%0.1 0.1 才与油2 90.7%98.2%89.9%97.2%88.2纠96.0%8:1.2%89.9%3 SN/T 4655-2016 A.1 参考条件1a)电离源方式:电喷雾电离;b)扫描方式:负离子模式;c)监测方式:多反应监测(MRM);d)干燥气温度(DGT):200 O(;e)干燥气流速(DGF):14L/mm;f)主主化器压力(GSl):20 PSl(氯气hg
16、)喃流气温度。GT):250 O(氮I();h)黯流气流速:11Lmin;i)离子眼喷雾电压IS:-2500 V;j)喷嘴电压:-2 000 V;附录A(资料性附录)参考质谱条件k)定性离子对(m/川、定量离子对(m/川，碎裂电压(V)，碰撞能量(eV)，保留时间见表A.lo表A.1多反应监测参数表1)组分名称定r!肖子对定LJ;肖子对驻留时间醉裂电)卡碰撞能LJ;保留时间川/zm/z ms(Frilg)/V(CE)/巳Vmm 167.S/63.0 100 380 25 草甘瞬167.8/81.0 167.8/63.0 100 380 1 1.68 167.8/124.。100 380 8 1
17、10.0/6:0.0 100 380 20 氨甲基瞬酸110.0/79.。110.0/63.0 100 380 35 4.20 110.0/81.0 100 380 10.2 参考条件2a)电离源方式:电喷雾电离;b)扫描方式:负离子模式;监测方式:多反应监测(MRM);d)碰撞气;在速比(CAD):4;e)气帘气压力(CUR):35;)雾化器压力(GSl):60Psi(氮气);1)非商业性声明:表A.1所列参考质谱条件是在Agilcnt6490 QQQ MS/MS型液质联用仪上完成的，此处列出试3命用仪?rg刑斗仪为捉供参考，并不涉及I句业口的，鼓胁标准使用者尝试不同厂家或*r的仪器。6 S
18、N/T 4655-2016 g)加热辅助气(币2):60 Psi(氮气kh)雾化温度:550(丁;i)离子游、喷雾电ffiIS:-4;-)00.0;j)定性离子对Cm/z)、定量离子对(rn/z)，去簇电压(V)，入口电压CV)，碰撞能量CeV)，碰撞?也出口电压(V)，保留时间见表1.2，表A.2多反应监测参数表2)定性离子对定量离子对驻留时间去簇电压人口电压碰撞能量碰撞池组分名称出口电压rn/z m/z ms 63.0000)Gly-AMPA-mix03-06.d-MRM CF=O.OOO DF=O.OOO(168.0000-81.0000)Gly-AMPA-mix03-06.d-MRM
19、CF=O.OOO DF=O.OOO(168.0000-150.0000)Gly-AMPA-mix03-06.d-MRM CF=O.OOO DF=O.OOO(110.0000-63.0000)Gly-AMPA-mix03-06.d-MRM CF=O.OOO DF=O.OOO(110.0000-79.0000)Gly-AMPA-mix03-06.d-MRM CF=O.OOO DF=O.OOO(110.0000-81.0000)Gly-AMPA-mix03-06.d k 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 Counts
20、vs.Acquisition Time(min)图B.1标准品多反应检测CMRM)色谱图CAgilent6 490 QQQ)从上至下以次为:167.8/63.0,167.8/81.0,167.8/150.0;11 0.0/63.0,11 0.0/79.0,11 0.0/8 1.0 SN/T 4655-2016 三;i;|11;j1;jj;jj|三，-三i二iiL二图B.2标准品多反应检测(MRM)色谱图(API4 000+QQQ)从上至下以次为:110.0/63.0,110.0/78.0,110.0/81.0;167.8/81.0,167.8/150.0,167.8/63.0,167.8/12
21、4.0 9 SN/T 4655-2016 Foreword This standard was drafted according to GB/T 1.1-2009.Please pay attention to some of the content of this document may be involved in the patent,the issu-ing authority of this document does not assume responsibility for the identification of these patents.This standard w
22、as proposed byand is under the charge of the Certification and Accreditation Admin-istration of the Peoples Republic of China.This standard was drafted by the Xiamen Entry-Exit Inspection and Ouarantine Bureau,Ilingbo Entry-Exit Inspection and Ouarantine Bureau and Fujian Entry-Exit Inspection and O
23、uarantine Bureau of the Peoples Republic of China.This main drafter of this standard is Xu Dunming,Chen Shubing,Wu Ming,Du Fengjun,Yu Yucheng,Liu Zhengcai,Zhang J ing,Zhang Zhigang.Note:This English version,a translation from the Chi门esetext,is solely for guidance.1 SNjT 4655-2016 Determination of g
24、lyphosate and its metablize residues in foodstuffs for export-HPLC-MS/MS method Scope This standard specifies the determination and confirmation of residues of glyphosate and its metablize residues Caminomethyl phosphonic acid)by HPLC-MSjMS in foodstuffs for export.This standard is applicable to the
25、 determination and confirmation of glyphosate and its metablize res-idues in tea,wheat.corn,rice,sugarcane,soybean,citrus,apple,peach,grape,banana,water-melon,ptisan and cottonseed oil for export.2 References The following documents were indispensable for the application of this standard.For dated r
26、efer-ences,only the dated edition was applied to this document.For undated references,the last edition of normative document(including all modification list)referred to applies.GBjT 6682 Water for analytical laboratory use-Specification and test methods 3 Principle The sample was extracted with wate
27、r and purify by dialysis bag,RP column and Graphitized carbon black,then detected bHPLC-MSjMS and quantified bexternal standard method.4 Reagents and Materials Unless otherwise specified,all the reagents used should be analyticallpure，water is 1 grade water stipulated bGBjT 6682.4.1 Methanol:HPLC gr
28、ade.4.2 Acetonitrile:HPLC grade.4.3 Ammonium acetate:HPLC grade.11 SN/T 4655-2016 4.4 Ammonium acetate solution(5 mmol/L,pH:10-11):Accurately weight 3.855 g(accurating to 0.000 1 g)ammonium acetate and dissolve with water to a volume of 50 mL,formulated as a ammo-nium acetate solution of 1 mol/L;The
29、n take 5 mL and dissolve with water to a volume of 1 000 mL with the pH adjusted to 10-11.4.5 Standard:glyphosate CGLY,CAS:1071-83-酌，Purity98%;aminomethyl phosphonic acid(AMPA,CAS:1066-51-酌，Purity主98%.4.6 Dialysis bag:diameter as 22 mm,semi-perimeter as 34 mm and molecular cut off as 3 500 Da.4.7 St
30、andard solution:Accurately weight GLY and AMPA 10 mg each and dissolve with water to a volume of 10 mL,formulated as a standard solution of 1 mol/L.4.8 Standard store solution:Accurately take any standard solution,dissolve with water to standard store solution of 10 用/mLof each.4.9 Standard working
31、solution:According to the concentration required,take a certain amount vol-ume of standard store solution and dissolve with water to appropriate concentration as standard working solution before use.4.10 Treatment for dialysis bag:10 cm-20 cm small fragments was cut by scissor,boiling water for 10 m
32、inutes,then cleaning bwater and stored in ethanol;Cleaning is necessarbefore and after use.4.11 OnGuard 11 RP column C 1.0 cc column):It was activated by 5 mL methanol and 5 mL water and let stand for half an hour before use;Clean with methanol after use.4.12 Graphitized carbon black CGCB):40m-60m，p
33、adding as the ProElut or equivalent.4.13 Membrane filter:0.22m.It is organic sstem and PTFE membrane.5 Apparatus and equipment 5.1 High Performance Liquid Chromatographwith triple quadrupole tandem mass spectrometer(HPLC-MS/MS)equipped with ESI source.5.2 Balance:sensibility reciprocal is 0.01 9 and
34、 0.000 1 9 respectivel.5.3 Tissues homogenizer.5.4 Grinder.12 SN/T 4655-2016 5.5 Homogenizer:15000 r/min.5.6 Centrifuge:5000 r/min above.5.7 High speed centrifuge:15000 r/min above.5.8 Ultrasonic water bath.5.9 Centrifuge tube:50 mL.5.10 Test tube:50 mL.5.11 Injection syringe.5.12 Vortex mixer.5.13
35、Pressured gas blowing concentrator.6 Sample preparat ion and storage 6.1 Sample preparation 6.1.1 Liquid samples Liquid samples were blended as backup.6.1.2 Fruit and vegetable samples The original sample is mixed from which 500 9 is taken for analysis.The samples were placed in a clean container to
36、 be used as the test sample after grinded thoroughly and the container having the test sample is well sealed and labeled.6.1.3 Food grains and tea samples The original samples were mixed from which a 500 9 was taken for analysis.The sample is divided to two potions equally after grinded thoroughly a
37、nd passed through a 20 mesh sieve.Then the sample is placed in a clean container to be used as the test sample.The container having the test sample is well sealed and labeled.6.2 Storage of samples AII the samples should be stored at-5 oC.13 SN/T 4655-2016 Note:In the sample operation and preparatio
38、n process,it should be prevent the pollution of samples and content changes of residues.7 Method of Determination 7.1 Extraction Weigh 4 9(Accurate to 0.01 g)of liquid samples,fruit and vegetable samples and food grains sam-ples into 50 mL centrifuge tube(5.9)and add 6 mL water.Mix well for 3 min by
39、 homogenizer(5.5)of 15000 r/min and then centrifuge(5.7)for 5 min by 15 000 r/min either.Water phase was collected and dissolve with water to a volume of 10 mL.Weigh 2 9(Accurate to 0.01 g)of tea samples into 50 mL centrifuge tube(5.9)and add 10 mL wa-ter.Mix well for 3 min by homogenizer(5.5)of 15
40、000 r/min and then centrifuge(5.7)for 5 min by 15000 r/min either.Water phase was collected and dissolve with water to a volume of 10 mL.7.2 Clean-up 10 mL extracting solution was injected into dialysis bag(4.10)which had been activated and then put the dialysis bag into 50 mL test tube,the tube was
41、 added 10 mL water before.Take out the dialysis bag after it was balanced for 1 h in ultrasonic water bath,5 mL raffinate from tube was injected into RP column binjector，refuse the first 2 mL solution wash-out from RP column(4.11)and collect the remaining 3 mL.Adding 20 mg GCB(4.12)and vortex oscill
42、ation for 2 minutes,then the mixed solu-tion centrifuged for 5 minutes under 15 000 r/min.Take 2 mL supernatant into a clean tube and dr nitrogen(5.13)at 60 C.The residue dissolved with water to 0.5 mL,pass through PTFE membrane and ready for LC-MS/MS determination.7.3 Determination 7.3.1 HPLC-MS/MS
43、 operation conditions HPLC-MS/MS operation conditions:a)Column:IIH2 P-50 20 column,150 mm x 2.0 mm(i.d.),5m or equivalent;b)Mobile phase:A:5 mmol/L ammonium acetate aqueous solution(ammonium hydroxide adjust pH to 10-11),B:acetonitrile;flow rate:0.25 mL/min,gradient elution program were showed in ta
44、ble 1;c)Column temperature:35 C;14 SNjT 4655-2016 d)Injection volume:10L;e)Source:ESI;f)Scanning mode:Negative;g)Monitoring mode:MRM;h)Other MS parameters:see Annex A.Table 1 Mobile phase gradient elution program Time/min Mobile phase A/%Mobile phase B/%。20.0 80.0 2.00 20.0 80.0 2.01 80.0 20.0 8.00
45、80.0 20.0 8.01 20.0 80.0 12.00 20.0 80.0 7.3.2 HPLC-MSjMS determination 7.3.2.1 Oualitative determination Under the above HPLC-MSjMS operating conditions,the ratio of the chromatographic retention time of analyte shall correspond to that of the calibration solution.The relative intensities of the de
46、tected ions of each analyte shall correspond to those of the calibration standard solution at comparable con-centrations,the allowed relative deviation of relative intensity is less than within the table 2 toler-ance,the same compound in sample must be confirmed.Table 2 Maximun permitted tolerances
47、for relative ion intensities while confirmation Relative ion intensities/%50%20%-50%10%-20%10%Permitted relative:t20%:t25%:t30%:t50%tolerances/%7.3.2.2 Ouantitative determination According to the estimated approximate concentration of target compounds in the sample solution,15 SNjT 4655-2016 select
48、the standard working solution of similar concentration to that of sample solution.The responses of target compounds in the standard working solution and the sample solution should be in the linear range of the instrumental detection.The standard working solution should be injected ran-domly in-betwe
49、en the injections of the sample solution of equal volume.Under the above HPLC-MSj MS operating condition,the retention time of GL Y and AMPA is about 4.68 min and 4.20 min each.HPLC-MSjMS multiple reaction monitoring chromatogram of GL Y and AMPA standard are shown re-spectively in the Annex B.7.4 R
50、eagent blank te5t The reagent blank test is taken the same complete analytical procedure applied without the test por-tion or using an equivalent amount of suitable solvent in place of the test portion.8 Calculation and expression of the result Calculate the concentration of GL Y and AMPA in samples