1、中华人民共和国出入境检验检疫行业标准SN/T 1982-2007 进出口食品中氟虫腊残留量检测方法气相色谱-质谱法Determination of fipronil residues in food for import and export GC-MS method 2007-08-06发布2008-03-01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 1982-2007 前言本标准附录A和附录B均为资料性附录。本标准由国家认证认可监督管理委员会提出并归口。本标准主要起草单位:中华人民共和国山东出人境检验检疫局、中华人民共和国湖南出人境检验检疫局、中华人民共和国江苏出入境检验检疫
2、局。本标准主要起草人:王建华、李杰、黄志强、沈崇缸、蔡发、孙忠松、王敬塘。本标准系首次发布的出人境检验检疫行业标准。I 1 范围进出口食品中氟虫膳残留量检测方法气相色谱-质谱法本标准规定了食品中氟虫腊残留量检测和制样、气相色谱-质谱检测和确证方法。SN/T 1982-2007 本标准适用于夜菜、藕、草莓、花生、鸡肉、猪肉、鳝鱼、蜂蜜、板栗、茶叶和酱油中氟虫腊残留量的测定和确证。2 方法提要试样经乙腊提取，以正己烧液液分配和初级次级胶(PSA)固相萃取柱净化，用气相色谱-负化学源质谱测定，外标法定量。3 试剂和材料除另有规定外，所用试剂均为分析纯，水为二次蒸锚水。3. 1 丙酬z残留级o3.2
3、乙睛:残留级。3.3 正己烧:残留级。3.4 氯化铀:6500C灼烧4h，置人干燥器中冷却，备用。3.5 丙酣十正己烧(3十7):取300mL丙隅，加入700mL正己烧，摇匀备用。3.6 氟虫腊标准品(Fipronil，C12 H4 C12 F6凡OS，CAS120067-37-3):纯度大于等于96.5%。3.7 氟虫腊标准榕液的配制:准确称取适量标准品，用少量的丙酣榕解，并以丙酣配制成浓度为1. 0 mg/mL的标准储备液。根据需要再用丙酣十正己炕(3.5)稀释成适当浓度的标准工作潜液。保存于一180C冰箱中。3.8 丙基乙二胶键合硅胶(PrimarySecondary amine，PSA
4、)固相萃取柱:500mg, 3 mL或相当者。4 仪器与设备4. 1 气相色谱质谱仪z配有负化学源。4.2 固相萃取装置。4.3 均质器。4.4 旋转蒸发器。4.5 氮气浓缩仪。4.6 具塞离心管:50mL、100mLo 4. 7 浓缩瓶:50mL、100mL。4.8 移液管:1mL、2mL、5mL、10mL。5 试样制备与保存5. 1 试样制备5. 1. 1 粮谷取有代表性样品量500g，用磨碎机全部磨碎并通过2.0mm圆孔筛。混匀，均分成两份作为试样，SN/T 1982-2007 分装入洁净的的容器内，密闭，标明标记。5. 1.2 水果或蔬菜取有代表性样品500g，将其可食用部分切碎后，依
5、次用食品捣碎机将样品加工成浆状。混匀，装入洁净的容器内，密闭，标明标记。5. 1. 3 肉及肉制品取有代表性样品500g，取可食部分经捣碎机充分捣碎均匀，装入洁净容器内作为试样。密闭，标明标记。5. 1.4 蜂蜜及蜂制晶取有代表性样品量500g，对无结晶的蜂蜜样品将其搅拌均匀;对有结晶析出的蜂蜜样品，在密闭情况下，将样品瓶置于不超过60C的水浴中温热，振荡，待样品全部融化后搅匀，迅速冷却至室温，在融化时应注意防止水分挥发。装入洁净的容器，密闭，标明标记。5. 1.5 茶叶取有代表性样品500g，用磨碎机全部磨碎并通过2.0mm圆孔筛。?昆匀，装入洁净的洁净容器内，密闭，标明标记。5. 1. 6
6、 坚果取有代表性样品500g，用磨碎机全部磨碎并通过20mm困孔筛。混匀，均分成两份作为试样，分装人洁净的洁净容器内，密闭，标明标记。5. 1. 7 水产品取有代表性样品500g，从所取样品中取出约1kg，取可食部分经捣碎机充分捣碎均匀，均分成两份，分别装入洁净容器内作为试样。密封并标明标记。5.2 试样保存粮谷类试样于OOC40C保存F水果和蔬菜及其他类试样于-180C以下冷冻保存。在抽样及制样的操作过程中，应防止样品受到污染或发生残留物含量的变化。6 j!iliJ定步骤6. 1 提取称取10g试样(精确至0.1g)于100mL具塞离心管中，加入10mL水，准确加人40mL乙腊(3.2) ，
7、用均质器高速匀浆提取2min(酱油和蜂蜜仅需剧烈振荡10min) ，再加入5g氧化铀(3.4)，剧烈振荡10min,3 OOOr/ min离心10mino 6.2 净化6.2. 1 液液分配净化取上层提取液20mL(菠菜、藕、草莓)或10mLC花生、鸡肉、猪肉、鳝鱼、蜂蜜、板栗、茶叶和酱油)转移至50mL具塞离心管(4.5)中，加入10mL正己烧(3.3)，振摇3min，静置分层，弃去上层正己烧相，再用10mL正己烧重复操作一次，弃去正己皖相F下层乙腊相收集于100mL浓缩瓶中，于400C水浴中浓缩至近干，加入1.0mL丙酣十正己皖(3.5)溶解残渣。6.2.2 圈相萃取(SPE)净化使用前用
8、5mL丙酣十正己烧(3.5)预淋PSA柱。将样液(6.2.1)倾人柱中，用10mL丙酣十正己烧(3.5)进行洗脱，控制流速小于等于2mL/mino收集全部洗脱液于50mL浓缩瓶中，于40.C水浴中浓缩至近干。用丙酣十正己烧(3.5)榕解并定容至1.0 mL，供气相色谱-质谱仪测定。6.3 测定6.3. 1 气相色谱-质谱条件a) 色谱柱:HP-5MS石英毛细管柱，30mXO. 25 mm(内径)，膜厚0.25m，或相当者;b) 色谱柱温度z初始温度为700C，以30oC/min程序升温至200oC，保持10min，再50.C/min 2 程序升温至270.C，保持4min; c) 进样口温度:
9、250.C;d) 色谱-质谱接口温度:280.C;e) 载气z氮气，纯度大于等于99.999%，恒压模式，柱头压1.45 MPa; f) 进样量:1L;g) 进样方式:无分流进样，0.65min后开阀ph) 电离方式:负化学电离pi) 离子源温度:150.C ; j) 四极杆温度:150.C; k) 反应气z甲:院，纯度大于等于99.99%; 1) 测定方式z选择离子监视j方式Fm) 选择监测离子(m/z):定量366，定性333、368、400;n) 溶剂延迟:4.0min。6.3.2 气相色谱-质谱检测及确证SN/T 1982-2007 根据样液中被测物含量情况，选定浓度相近的标准工作榕液
10、，对标准工作溶液与样液等体积参插进样测定，标准工作溶液和待测样液中氟虫腊的响应值均应在仪器检测的线性范围内。如果样液与标准工作榕液的选择离子色谱图中，在相同保留时间有色谱峰出现，并且在扣除背景后的样品质谱图中，所选离子均出现，所选择离子的丰度比与标准品对应离子的丰度比，其值在允许范围内(允许范围见表1)。在6.3. 1条件下，氟虫腊保留时间是11.1 mn，其监测离子(m/z)为m/z366、333、368、400(其丰度比为100: 27 : 70 : 35)对其进行确证;根据定量离子m/z366对其进行外标法定量。在6.3.1条件下，氟虫睛标准物的气相色谱-质谱总离子流色谱图和全扫描质谱图
11、参见附录A中图A.1和附录B中图B.l.表1使用定性气相色谱质谱时相对离子丰度最大容许误差相对丰度(基峰)/%GC产MS/NCI相对离子丰度最大允许误差/%6.4 结果计算和表述50 土202050 :1: 25 用色谱数据处理机或按式。)计算试样中氟虫睛残留量z式中zx=兰主兰主-hs Xm x-一一试样中氟虫腊残留量，单位为毫克每千克(mg/kg); h一一样液中氟虫腊的色谱峰高，单位为毫米(mm);hs一一标准工作液中氟虫腊的色谱峰高，单位为毫米(mm);c-一标准工作液中氟虫腊的浓度，单位为微克每毫升(g/mL); V一-样液最终定容体积，单位为毫升(mL); m一一最终样液所代表的试
12、样质量，单位为克(g)。7 测定低限、回收率7. 1 测定低限本方法的测定低限为0.002mg/峙。7.2 回收率样品的添加浓度及回收率的实验数据见表201020 士30(10 土50( 1 ) 3 SN/T 1982-2007 表2样晶的添加浓度及回收率的实验数据样品添加浓度/(g/kg)回收率范围/%样品添加浓度/(g/kg)回收率范围/%2 90. 01l0.。2 90. 01l0.。渡菜4 95. 0107. 5 鳝鱼4 92. 5107. 5 10 97. 01l2.。10 97. 0119.。2 95. 01l0.。2 90. 0110.。藕4 97. 5110.。蜂蜜4 92.
13、5107. 5 10 96. 01l2.。10 96. 0112.。2 81. 5100. 8 2 95. 0115.。草莓4 92. 5107. 5 板栗4 92. 5107. 5 10 96. 01l2.。10 95. 0114.。2 90. 01l0.。2 90. 0110.。花生4 90. 01l2. 5 茶叶4 97. 5112. 5 10 94. 01l7.。10 97. 0113.。2 90. 01l0.。2 95. 0110.。鸡肉4 95. 0107. 5 酱油4 92. 5115.。10 94. 0112.。10 94. O115.。2 90. O 110.0 猪肉4 92
14、. 5112. 5 10 94. 0112.。4 SN/T 1982-2007 附录A(资料性附录)氟虫睛标准物质的气相色谱-盾谱总离子流色i普圄Abundance TIC: 10PPB.D 11. 15 AnUAUAUAUAUnununU内UAUAUnunAAUnununnununuvnvnunvnHunHMAUnHVAHunununuAHvnvnHVAHVAHUOZHvnHVAHvn aazo4nunonoaaz04AUOMUPOAUZ04oenbaa-nanuoooa绪。，aazaaaaxnO句。uqoqon404qz。4。4且且且且i._啕曲帽.，._-啕-呵严-5.00 6.00 7
15、.00 8.00 9.00 10.0011.00 12.00 13.00 14.0015.0016.0017.0018.0019.00 min Time-圈A.1氟虫腊标准物的气相色谱质谱总离子流色i普固附录B(资料性附录)氟虫睛标准物质的气相色谱质谱圈Abundance Scan 850 (11.202 min) :FIPRO. D 366 s n 。哼，nwd nd 4I 。0un4 A哇iz nunUnunUUnunu内Un白nununAUnunUUAUAAUnnHvnHuwaHuwhHVaHvnHwnuwnuvhHUAHVHVAHvnHunuw向HuhHV向HVAHvnHvnHW内HV
16、nunAUnununununnUAUnunununununUAUAUn向UnununununununU向U向nunununnU向AUnunnnunununanununununUAUnnunununuUnnnununununuinunwuonu句，PO俨Oaazqdn4Anun臼06COFhaazn004in404411AASA-A喃自AtA1A喃自A331 400 rn/z_ 280 290 300 310 320 330 340 350 360 370 380 390 400 图B.1氟虫睛标准物质气相色谱，质谱固5 SN/T 1982一2007Foreword Annex A and An
17、nex B of this standard are informative annexes. This standard was proposed by and is under the charge of the Certification and Accreditation Admin stration of the People s Republic of China. This standard was drafted by the Shandong Entry-Exit Inspection and Ouarantine Bureau、HunanEn try-Exit Inspec
18、tion and Ouarantine Bureau and Jiangsu Extry-Exit Inspection and Ouarantine Bureau of the People s Republic of China. This main drafters of this standard are: Wang Jianhua, Li Jie, Huang Zhi qiang , Shen Cheng yu , Caifa Sun zhongsong Wang Jingtang This standard is a professional one which is promul
19、gated for the first time. Note: This English version , a translation from the Chinese text, is solely for guidance. 6 SN/T 1982-2007 1 Scope Determination of fipronil residues in food for import and export-GC-MS method This standard specifies the method of sample preparation and determination of fip
20、ronil residue in foodstuffs bgas chromatography-mass spectrometry. This standard is applicable to the determination and confirmation of fipronil residue in spinach , lotus, strawber时，peanut，chicken , pork, ling , honey, chestnut, tea and soy. 2 Principle The test sample is extracted with acetonitril
21、e, then the extract is partitioned with n-hexane before cleaning up procedure bpassing through a PSA solid phase extraction (SPE) column. The deter mined by GC-NCI-MS , and quantitated bexternal standard method. 3 Reagents and materials AII the reagents used should be analytically pure unless otherw
22、ise specified. Water is redistilled water. 3.1 Acetone: residual grade. 3.2 Acetonitrile: residual grade. 3.3 n-Hexane. residual grade. 3.4 Sodium chloride:heated at 6500C for 4 h,and stored in a sealed container. 3.5 Acetone-n-Hexane (3 + 7) : Dilute 300 mL acetone with n-Hexane to the volume of 1
23、000 mL. 3.6 Fipronil standard (Fipronil，C12比CI2F6N40S,CAS 120067-37-3): Purit96.5%. 3.7 Standard stock solution:Accurately weigh appropriate amount of fipronil standard and dissolve with a little volume of a臼tonefollowed by a further dilution to the final ncent阳tionof 1. 0 mg/mL. Then dilute the sta
24、ndard stock solution with acetone to make standard working solution. of required con-7 SN/T 1982-2007 centration and stored at - 18 .C. 3.8 SPE column:500 mg ,3 mL or equivalent. 4 Apparatus and equipment 4. 1 Gas chromatographequipped with negative chemical ionization mass spectrometry. 4.2 SP巳12GC
25、olumn Processor. 4.3 Homogenizer. 4. 4 Rotary vacuum evaporator. 4. 5 Nitrogen evaporator. 4.6 Centrifuge tube:50 mL,100 mL with stopper. 4.7 Concentrating bottle:50 mL, 100 mL. 4.8 Graduated pipettes: 1 mL,2 mL,5 mL,10 mL. 5 Preparation and storage of test sample 5. 1 Preparation of test回mple5. 1.
26、1 Cereals Take approximately 500 9 of representative sample. Grind with a grinder to pass through a 2.0 mm round-hole sieve. Mix thoroughly and divide into two equal portions. Each portion is placed into a clean container,as test sample,sealed and labelled. 5.1.2 Fruits and vegetables Take approxima
27、tely 500 9 of representative sample(without wash by water). The edible parts are blended and homogenized in a high speed blender. Divide into two equal portions. Each portion is placed into a clean container as test sample,sealed and labelled. 5. 1. 3 Meat and meat product Take approximately 500 9 o
28、f representative sample. The edible parts are blended and homogenized in 8 SN/T 1982-2007 a high speed bJender. Oivide into two equaJ portions. Each portion is pJaced into a cJean container as the test sampJe ,seaJed and JabeJed. 5. 1. 4 Honey and honey product Take approximateJ500 9 of representati
29、ve sample. Homoenize the non- crystallization honey and the crystallized honey in the sample bottle should be warmed under water bath at the temperature of no more than 60oC. Shake the bottel until the sample is completely dissolved. Sample should be homoge nized and cooled down to room temperature
30、rapidly. 00 prevent the evaporating of water during the heating procedure. Then place into a clean container as the test sample,sealed and labeled. 5.1.5 Tea Take approximately 500 9 of representative sample. Grind and pass through a 2. 0 mm round-hole sieve. Mix thoroughly and place into a clean co
31、ntainer as the test sample,sealed and labeled. 5.1.6 Nut Take approximately 500 9 of representative sample, Grind and pass through a 2. 0 mm round自holesieve. Mix thoroughly and place into a clean container as the test sample,sealed and labeled. 5. 1. 7 Aquatic product Take approximately 500 9 of rep
32、resentative sample. The edible parts are blended and homogenized in a high speed blender. Oivide into two equal portions. Each portion is placed into a clean container as the test sample. sealed and labeled. 5. 2 Storage of test sample The test samples of cereals and oil seeds should be stored at th
33、e range of OoC _40C. The test samples of fruits and vegetables should be stored below - 180C. Ouring sampling and sample preparation. pre圄caution should be taken to avoid contamination or anfactors which may cause the change of residue content. 6 Procedure 6.丁ExtractionWeigh ca. 10 9 of the te8t sam
34、ple into a 100 mL centrifuge tube equipped with a stopper. And accu rately add 10 mL of water,40mL acetonitrile(3. 2) into the flask. Extract for 2 min in a high speed ho mogenizer(shake for 10 min for sauce and honey only). Add 5 9 Sodium chloride (3.4) , shake for 10 min, centrifuge for 10 min at
35、3 000 r/min. 9 SN/T 1982一20076. 2 Cleaning up 6. 2. 1 Liquid/liquid partition Transfer the 20 mL (spinach, lotus, strawberry) or 10 mL (peanut, chicken , pork, ling , honey, chest nut, tea and sauce) of supematant into a 50 mL centrifuge tube(4.日，add10 mL of hexane ,shake for 3 min and place aside f
36、or separation. Discard the hexane phase. Repeat above procedure and condense acetonitrile to nearly dryness bya rotary evaporator at 40 t. Dissolve the residue with 1.0 mL of ac etone-hexane (3.5) for SPE purification. 6. 2. 2 SPE cleaning up Rinse a PSA column with 5 mL of acetone-hexane (3.5). Loa
37、d the above solution to column. Elute the column with 10 mL of acetone-hexane (3.日，andflow rate below 2 mL/min. all eluates are transfered into a 50 mL concentrate bottle and evaporated to dryness by a rotary evaporator at 40 t. Dissolve the residue with acetone-hexane(3. 5) to exact volume of 1. 0
38、mL for determination bGC-MS. 6. 3 Determination 6. 3. 1 GC-MS operatingndition a) Chromatographic column: HP-5MS silica capillary column 30 m X O. 25 mm (i. d. ) ,0. 25m film thickness,and or equivalent; b) Column temperature: 70oC , ramp at 30oC/min to 200oC , hold for 10 min, and then increase at
39、50t/min to 270t ,hold for 4 min; c) Injector temperature: 250.C ; d) Interface temperature:280t ; e) Carrier gas: Helium, purity;:?:99. 999 %, constant pressure mode: 1. 45 MPa; f) Injection volume: 1L; g) Injection mode Splitless,open the valve after O. 65 min; h) Electrical ionization mode: NCI; i
40、) Quadropole temperature: 150.C ; j) lon source temperature: 1500C ; 10 SN!T 1982-2007 k) Methane二三99.99%;1) Selected ion monitoring mode; m) Monitoring ions (m!z) :quantitied by 366 ,qualified by 333 ,368 ,400; n) Solvent dela:4.0 min. 6. 3. 2 Quantitation and qualification by GC-MS Select appropri
41、ate standard working solution with similar concentration level to that in sample solu tion, The standard working solution should be injected before and between the injections of the sam ple solutions with same injection volume. The response value of fipronil in the standard working solu tion and sam
42、ple solution should be within the linear range of the instrumental detection. Permitted tolerance for similarity of relative abundance ratio is listed as table 1. When retention time of target peak is according with that of standard solution,postitive samle will be proved based on 四lected monitoring
43、 ions (m!z) 366 ,333 ,368 ,400 (relative abundance ratio: 100 : 27 : 70 : 3日，with(m!z) 366 for quantitation. Under chromatographic condition above(6. 3. 1) , rention time of fipronil standard is 11. 1 min , GC-MS selected ion chromatogram and mass spectrum of the fipronil standard are shown respecti
44、vely as figure A. 1 in annex A and figure B. 1 in annex B. Table 1-Maximum permitted tolerance for relative ion intensities using a range of mass spectrometric techniques Relative intensity(base peak)/% GC-MS/NCI (relative) /% 50 士206. 4 Calculation and expression of the result 20-50 士2510-20 土3010
45、士50Calculate the content of fipronil residue in the test sample by GC-MS data processor or according to the followed formula(1): h x c x V x= . ( 1 ) hs x m where X -the residue content of fiproni I in the test sample, mg!kg ; h-the peak height of fipronil in the sample solution,mm; hs一thepeak heigh
46、t of fipronil in the standard working solution ,mm; 11 SN/T 1982-2007 C一theconcentration of fipronil in the standard working solution，阅/mL;V-the final volume of the sample solution,mL; m-the corresponding mass of the test sample representing the final sample solution,g. 7 Limit of determination and
47、revery 7. 1 Limit of determination The limit of determination of this method is 0.002 mg/kg. 7. 2 Recovery The experimental data of the concentrations of fipronil in the fortified sample and its corresponding recoveries are ,seen in table 2. sample spinach lotus strawberry peanut chicken pork 12 Tab
48、le 2-The experimental data of the concentrations of fipronil in the fortified sample and its corresponding recoveries Added concentrations/ Re，very range/ sample Added concentrations/ Recovery range/ (g/同)% (g/kg) % 2 90.0-110.。2 90.0-110.。4 95.0-107.5 ling 4 92.5-107.5 10 97.0-112.。10 97.0-119.。2 9
49、5.0-110.。2 90.0-110.。4 97.5-110.。honey 4 92.5-107.5 10 96.0-112.。10 96.0-112.。2 81.5-100.8 2 95.0.115.。4 92.5.107.5 chestnut 4 92.5-107.5 10 96.0-112.。10 95.0-114.。2 90.0-110.。2 90.0.110.。4 90.0-112.5 tea 4 97.5-112.5 10 94.0.117.。10 97.0-113.。2 90.0.110.。2 95.0.110.。4 95.0-107.5 soy 4 92.5.115.。10 94.0.112.。10 94.0.115.。2 90.0-
