1、 必言-一-、i.:i:AlI_.o 中华人民共和国出入境检验检疫行业标准SN/T 1973-2007 进出口食品中阿维菌素残留量的检测方法高效液相色谱一质谱/质谱法Determination of abamecin residues in food for import and export HPLC-MS/MS method 2007-08-06发布2008-03-01实施豆豆霞飞酬献甜ti.lli访而飞杠F中华人民共和国发布国家质量监督检验检疲总局SN/T 1973-2007 前言本标准的附录A、附录B均为资料性附录。本标准出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河
2、北出入挠检验检疫局、中华人民共和国辽宁出入挠检验检疫局、中华人民共和国福建出入统检验检疫局、中华人民共和国山东出人境检验检疫局、中国检验检疫科学研究院负责起草。本标准主要起草人z玉凤池、林维宣、马育松、郭春海、陈瑞春、李丽、杨芳、李淑娟。本标准系首次发布的出入境检验检疫行业标准。I 1 范围进出口食品中阿维菌素残留量的检测方法高效1在相色谱-质i昔/质谱法本标准规定了食品中阿维菌素残臼量的高效液相色谱质谱/质i苦检测方法。SN/T 1973-2007 本标准适用于大米、大蒜、菠菜、苹果、板栗、茶叶、牛肉、羊肉、鸡肉、鱼肉、赤苟、食自由和蜂蜜中阿维2 方法提要I ! 蜂蜜和食附样品先用反革睹了t
3、r-:IH怦取柱净化2时也样品用乙脐提取，用中性氧化铝固相萃取柱净化，高效液相色谱质i苦/质谱祖国外标法定量f3 试剂和材料/ / 除另有规定外，试剂均万升忻纯，水为主IT!ft!巧穴T18 Mfl)lo 3. 1 乙脂2高效液相色谱纯叫二i 3.2 乙酸水溶液(O.l%)，!ft1 mL乙自主，以水定容至1000mLl 3.3 乙脂水溶液s乙腊+水(1+6.体积比)。度的标准工作液。保存于4C冰箱内。4 仪器和设备4. 1 高效液相色谱质谱/质谱仪，配有大气压化学电离源(APCI源)。4.2 均质器。4.3 离心机.3000r/min以上。4.4 涡旋振荡器。4.5 固相萃取装置。4.6 旋
4、转蒸发器。4. 7 氮吹仪.SN/T 1973-2007 5 试样的制备与保存5. 1 苹果、大蒜、菠菜、板栗苹果、板栗E去皮或核，取可食部分约500g，用捣碎机捣碎，装入洁净容器作为试样.密封并做好标识，于4C冰箱内保存。大蒜、菠菜z取样品约500g，用捣碎机捣碎，装人洁净容器作为试样，密封并做好标识，于4C冰箱内保存。5.2 大米、茶叶、赤巧、食醋取样品约500g fll粉碎饥粉碎至全部通过20日筛，装入洁净容器作为试样，密封并做好标识，室温保存。食黯可装入洁净容器，密封并做好标识，室温保齐。5.3 牛肉、羊肉、鸡肉、鱼肉取样品中有代表性的可食部分约500g，用捣碎机捣碎，装入洁净容器作为
5、试样，密封并做好标识，于ISC冰箱内保存。5.4 蜂蜜取有代表性样品约500g，未结晶样品将其用力搅拌均匀，有结晶析出样品可将样品瓶盖塞紧后，置于不超过60C的水浴中，待样品全部溶化后搅匀，迅速冷却至室温。由备好的样品装人i古净容器内密封并做好标识，室温保存。5.5 要求制样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 测定步骤6. 1 提取6. 1. 1 大蒜、菠菜、苹果、饭票、牛肉、羊肉、鸡肉、鱼肉样品准确称取5g(精确至0.01g)均匀试样，加入5g元水硫酸纳和15mL乙腑，以10000 r/min均质2 min , 3 000 r/min下离心5min.上;青液经元水疏般的过
6、滤并转人浓缩瓶中。用10mL乙腊再提取一次，合并提取液。将提取液于40C水浴下浓缩至2mL3 mL。6. 1. 2 大米、茶叶、赤苟样品准确称取5g(精确至0.01g)均匀试样，用2mL水将试样润湿(茶叶用20mL 7(润湿)，放置15 min，加人5g元水硫酸纳和15mL乙脂，以10000 r/min均质2min, 3 000 r/min下离心5min，将上清液转人浓缩瓶中。用10mL乙肪再提取一次，合并提取液。将提取液于40C水浴下浓缩至2 mL3 mL。6.1.3 食醋、蜂蜜样品准确称取5g(精确至0.01g)均匀试样，加入30mL水，涡旋i昆匀。6.2 净化6.2. 1 大米、大蒜、菠
7、菜、苹果、板栗、茶叶、牛肉、羊肉、鸡肉、鱼肉、赤苟样品用3mL乙脂对中性氧化铝柱进行预淋洗。将6.1.1和6.1.2得到的样品提取液转入中性氧化铝柱，用5mL乙H百分两次洗涤浓缩瓶并将洗涤液转人中性氧化铝住中，调整流速在1.5 mL/min左右，用2 mL乙脯淋洗小柱，收集全部流出液。将流出液在50C下吹干，用1.00 mL乙脂溶解残渣，滤JW;过滤，但飞液相色谱-质谱/质i昔测定。6.2.2 食醋、蜂蜜样品依次用5mL乙睛和5mL乙脂水溶液j(，tC8回相萃取柱进行预淋洗。将6.1.3得到的样品稀释液加入C18固相萃取柱，调整流速在1.5 mL/min左右，加入10mL水淋洗CI8因相萃取柱
8、，将回相萃取柱SN/T 1973-2007 吹干。加入5mL乙肪进行洗脱，收集全部洗脱液。将洗脱液在50C下吹干，用1.00 mL乙脂溶解残渣，滤膜过滤，供液相色i吉-质谱/质i苦测定。6.3 测定6.3. 1 液相色i昔条件6.3. 1. 1 色消柱:C18柱.150mmX 2.1 mm(内径)，粒度5m.6.3. 1. 2 流动相z乙脂+乙酸水溶液(0.1%)70+30. 6.3. 1. 3 流速:0.3mL/min. 6.3. 1. 4柱1riI:40C。6. 3. 1. 5 进样量:20L.6.3.2 质i普条件6.3.2. 1 离子源z大气压化学电离源(APCI源)，负离子监视l模式
9、。6.3.2.2 质谱仪参数参见附录A。6.3.2.3 监lJ!tl离子对(m/z):定性离子对(872/565，872/854)，定量离子对(872/565)。6.3.3 高效液相色i营质谱/质谱测定根据试样中阿维菌素的含量情况，选择浓度相近的标准工作液进行色谱分析，以峰面积按外标法定量。在上述包谱条件下，阿维因素的参考保留时间为11.3 min，标准溶液的选择离子流图参见附录B中图B.1、图B.2。6.3.4 确证按照上述条件测定样品和标准工作液，如果检测的质量包谱岭保留时间与标准工作液一致;定性离子对的相对丰度与相当浓度的标准工作浓的相对丰度一致，相对丰度偏差不超过表1的规定，则可判断样
10、品中存在相应的被测物。标准溶液的二级质i古图参见附录B中图B.3.表1定性确证时相对离子丰度的最大允许偏差相对离子丰度/%允许的相对偏差/%6.3.5 空白试验50 士20除不加试样外，均按上述操作步骤进行。7 结果计算与表述2050 士251020 土30按数据处理软件处理或式()计算样品中阿维菌素残留量，计算结果需扣除空白值。式中gXA.cV As m X 试样中阿维菌素残臼茧，单位为毫克每千克(mg/kg); c一一阿维菌素标准工作液的浓度，单位为微克每毫升(g/mLl; V一一样品鼓终完容体积，单位为毫升(时.); A-一样液中阿维菌素的峰面积;As-阿维因素标准工作液的峰面积gm 最
11、终样液代表的试样质量，单位为克(g)。8 il!tl定低限和回收率8. 1 测定低限本方法中阿tlE菌素的测定低限为O.005 mg/kg, :;:;10 土50( 1 ) 3 SN/T 1973-2007 8.2 回收率样品的添加浓度及回收率的实验数据见表2.样品名称大米大蒜菠菜苹果板栗茶叶牛肉羊肉鸡肉鱼肉蜂蜜赤苟食醋4 表2样品的添加浓度及回收率的实验数据添加浓度!(mg!kg)0.005 0.01 67.2%-99.2% 74.6%-90.8% 61. 6%-85. 2% 63.2%-83.4% 68.0%-106.8% 79.8%-99.6% 69.2%-100.8% 76.6%-10
12、7.6% 78.4%-111. 2% 86.4% -102.6.% 69.2%-94.。 74.8%-85.8% 61. 2%-95. 6 问74.6%-92.2% 66. 0%-76. 8-:_F 79%-88.4% / 64.4%-85.6% /hpi96.oy 73.2%-96.8% 阿71. 6%飞到3.6%川%-9口%/7弘890-102.2%64.8%-85.2; 74. 6%-87. 0% 67.2%-74.4 65.6%-87. S% L_一一一一一一一一】中一Y J夕0.05 70.0%-86.6% 72.3%-84.9% 81. 7%-96.1% 73.1%-91. 7%
13、72.6%-83.6% 69.6%-90.6% 74.1%-99.1% 68.3%-79.7% 80.4%-101. 3% 85.2%-97.2% 66.6%-87.3% 70.9%-96.9% 76.2%-85.3% A.1 喷雾压力，60psio A.2 干燥气体流量:5L!min。A.3 干煤气体温度，350C。A.4 大气压化学电离源蒸发温度:400C。A.5 电晕电流，10000 nAo A.6 毛细管电压:3500Vo 附录A(资料性附录)质谱仪参数1)SN!T 1973-2007 1)非商业性声明.所列参数是在美国AgilentLC-MSD-Trap-VL离于阱质谱仪上完成的，此
14、处列出试验用仪器型号仅是为了提供参考，并不涉及商业目的，鼓励标准使用者尝试采用不同厂家或型号的仪器.5 SN/T 1973-2007 Intens 1500 1000 500 。2 图B.1Intens 150 100 50 。图B.2Intens 80 60 40 455 485 。450 500 6 附录B(资料性附录)标准溶液的选择离子流图和二级质谱图4 6 8 11. 3mm -565.3 1llZ 10 阿维菌素标准物质(10ng/mL)的选择离子流图(565m/z) 8 8 10 阿维菌素标准物质(10ng/mL)的选择窝子流图(854m/z) 565.3 789.5 Time(m
15、in) Time(min) 871. 5 8息3.576 1. 5 826.5 550 600 650 700 750 800 850 900 图B.3阿维菌素标准溶液的二级质i昔图SN/T 1973-2007 Foreword Annex A and annex B of this standard are informative annex This standard was proposed by and is under the charge of the Certification and Accreditation Admin istration of the Peoples Re
16、public of China This standard was drafted by Hebei Entry-Exit Inspection and Ouarantine Bureau of P. R. C, Liaoning Entry-Exit Inspection and Ouarantine Bureau of P. R. C, Fujian Entry-Exit Inspection nd Ouarantine Bureau of P. R. C, Shandong Entry-Exit Inspection and Ouarantine Bureau of P. R. C, C
17、hinese Acade my of Inspection and Ouarantine. The standard was mainly drafted by Wangfengchi , Linweixuan, Mayusong , Guochunhai , Chenrui chun , Li li , Yangfang and Lishujuan This standard is a professional standard for entry-exit inspection and quarantine peomulgated for the first time. 7 SN/T 19
18、73-2007 Determination of abamectin residues in food for import and export HPLC-MSjMS method 1 Scope This standard specifies the method 0f the determination of abamectin residues in food by LC-MS/MS. - -a This standard is applicable to the deterjnination of abamectin 陪siduesin r,ice. garlic. spinach.
19、 apple. chestnut. tea. beef. mutton. chicken. fish. chishao. yit1egar and hOney samples. 2 Principle / / /-Honey and vinegar samples were dilutd with water and cleaned up with!C18 column. other samples were extracted with acetonitrile and 申!eanedup with LC-Alumnum-N clumn. Determined by LC MS/MS. us
20、ing external standard meth萨a.-一一一一一)甲一-牛一-一3 Reagents and materials Unless otherwise specified. all reag (18 Mn). 3. 1 Acetonitrile, HPLC grade. 3.2 Acetic acid solution (0. 1 %), Pipelt1 mL of actic acicf-and cjilte to 1 000 mL with water 飞、飞_-_/1 3.3 Acetonitrile solution: Acetonitrile+ water(1 +
21、6. V / V). 3.4 Anhydrous sodiurn sulphate: Ignite for 4 h at 650C cool to room temperature in desiccator and keep in a tightly closed container. 3.5 LC-Aluminurn-N cartridge: 1 000 mg. 3 mL. 3.6 C18 cartridge:1 000 mg. 6 mL. 3. 7 Membranes for organic: O. 45m 8 SN/T 1973-2007 3.8 Abamectin standard:
22、 (C.H72014 CAS NO: 71751-41.刀.contentof abamectin Bla 87%. fol lowing abamectin content is calculate with abamectin Bla. 3.9 Abamectin standard stock solution: Accurately weigh approriate abamectin standard. dissolve e and quantitatively with acetonitril. the concentration of solution is 100g/mL. Th
23、e stock solution should be stored at -18C refrigeratory. 3.10 Abamectin standard middle solution: Pipette the above stock solution and dilute with acetoni trile. the concentration of solution is 1g/mL. The solution should be stored at 4C refrigeratory. 3. 11 Abamectin standard working fclu召O:Pipette
24、 appropriate volur加ofthe above standard solu tion and dilute to proposed concentrtion with acetonitrile. The workit电solutionshould be stored at 4C refrigeratory. I I 4 Apparatus and equipment ./丁/, 4.1 Li阳i问q刷c仙hroma叫gr即hycom附bi哼中dw怕a创tomosp附tr(毡3盯1etry.4.2 Homogenizer. 4.3 Centrifuge. 3000 r/min. 4
25、.4 4.5 4.6 阳?UJRotary evaporator.飞f4.7 N, evaporator. 飞、4.p/5 Sample preparation and storage 5.1 App怡.garlic. spinach and chestnut / Apple and chestnut: Peel or stone is discard. Take the representative portions from the whole sam ple. It is about 500 9 and grind in a blender. Keep the prepared samp
26、le into a clean container. seal and label. The test sample is stored in 4C refrigeratory Garlic and s口inach:The sample is about 500 9 and grind in a blender. Keep the prepared sample into 9 SN/T 1973-2007 a clean container. seal and label. The test sample is stored in 4C refrigeratory. 5.2 Rice. tea
27、. chishao and vinegar The sample is about 500日.grind thoroughly and let pass through a 20 mesh sieve. Keep the prepared sample into a clean container. seal and label. The sample is stored in room temperature. Vinegar was kept into a clean container. seal and label. The sample is stored in room tempe
28、rature. 5.3 Beef. mutton. chicken and fish Take the representative edibility portions from the whole sample. It is about 500 9 and grind in a blender. Keep the prepared sample into a clean container. seal and label. The test sample is stored in - 18C refrigeratory. 5. 4 Honey samples Take the repres
29、entative portions from the whole sample. It is about 500 g. The sample which is not crystallized shall be stirred well to make homogeneous. If the sample is crystallized. it must be warmed in a water-bath below 60C with the sample bottle covered tightl. mix thoroughly when all sample has melted. the
30、n cool immediately to room temperature. Keep the prepared sample into a clean container. seal and labe l. Honey sample is stored in room temperature. 5.5 Requirement In the course of sample preparation. pre臼utionsshould be taken to avoid contamination or any factors which may臼usethe change of residu
31、e content. 6 Procedure 6. 1 Extraction 6.1.1 Garlic. spinach. apple. chestnut. beef. mutton. chicken. fish samples Weigh ca 5 9 (accurate to O. 01 g) of the test sample. add 5 9 anhydrous sodium sulphate and 15 mL acetonitrile. Homogenize for 2 min at 10 000 r/min and centrifuge at 3 000 r/min for 5
32、 min. The su pernatant layer is filtrated by anhydrous sodium sulphate and transferred to a flask. Repeat the ex traction of the residue in the same way with 10 mL acetonitrile and combined the solution. Evaporate to about 2 mL-3 mL in a water bath below 40 C. 10 S/T 1973-2007 6.1.2 Rice , tea and c
33、hishao samples Weigh ca 5日(accurate to O. 01 g) of the test sample , the test sample is wetted with 2 mL water (tea:20 mL), then add 5 9 anhydrous sodium sulphate and 15 mL acetonitrile. Homogenize for 2 min at 10000 r/min and centrifuge at 3 000 r/min for 5 min. The supernatant layer is transferred
34、 to a flask. Repeat the extraction of the residue in the same way with 10 mL acetonitrile and combined the solution. Evaporate to abot 2 mL-3 mL in a water bath below 40:. 6. 1.3 Honey and vinegar sample Weigh ca 5 9 (accurate to O. 01日)of the test sample. add 30 mL water and mix the solution. 6.2 C
35、lean up 6.2.1 Rice , garlic, spinach , apple, chestnut , tea , beef, mutton, chicken , fish and chishao sam ples Condition the LC-Aluminum-N cartridge with 3 mL acetonitrile. Transfer the concentrated solution into the column , the flow rate was adjusted to about 1.5 mL/min. eluted the column with 2
36、 mL ace tonitrile. Collect al1 the eluted solution. Evaporate the solution just to dryness under a stream of dry nitrogen using a heating block at 50:. Dissolve the residue with 1.00 mL acetonitrile and filtrate by membrane. The solution is ready for determination. 6. 2. 2 Honey and vinegar sample C
37、ondition the C cartridge with 5 mL acetonitrile and 5 mL acetonitrile solution. Transfer the solu tion into the column. the flow rate was adjusted to about 1.5 mL/min. wash the column with 10 mL water and dry the column with air. eluted the column with 5 mL acetonitrile. Collect the eluted solu tion
38、. Evaporate the solution just to dryness under a stream of dry nitrogen using a heating block at 50:. Dissolve the residue with 1.00 mL acetonitrile and filtrate by membrane. The solution is ready for determination. 6. 3 Determination 6.3. 1 HPLC parameters 6.3.1.1 Column: C. 150 mm x 2.1 mm(i. d. )
39、.5m 6.3.1.2 Mobile phase: acetonitrile+ acetic acid solution(O. 1 %) = 70 + 30. 6.3.1.3 Flow rate: 0.3 mL/min. 6.3.1.4 Column temperature: 40C. 11 SN/T 1973-2007 6.3.1.5 Injection volume, 20L 6.3.2 Mass spectrometry parameters 6.3.2. 1 Source, atomospheric pressure chemical ionization (APCD , negati
40、ve mode. 6.3.2.2 See mass spectrometry parameters in annex A 6.3.2.3 Qualitative ions(m/z) , qualitative ions(872/565, 872/854) , quantitative ions (872/565). 6. 3. 3 HPLC-MS/MS deterl 川叫怡协Y盯刚川阶问叫川叫平呻叫叫咀d们叫f.A川A刮!i怕n川、川i附叩r刷kin咱9solution which has a similar coc坦tr6t盹G 叫f l a i t w l t h m a k m a by
41、 M 叫stand 时-Under the above chromatography c咱们rton，the retention time of abamectin is 11.3 min. Fo门he./ , chromatogram of standard are shgfn in figure 8:1 and 8.2 in annex 8. f 卢J6.3.4 Confirmation of LC-IIS/MS I Under above conditio阳，the!.Stndao:d.worling.solution-and sample solution is injected. I
42、f the reten-Relative ion intensities/% Permitted tolerances/% 6. 3. 5 81ank test :t 30 10 :t 50 The operation of the blank test is the same as the described in the method of determination , but with the omission of sample addition. 7 Calculation and expression of the result The calculation of the co
43、ntent of abamectin in the test sample is carrid out by data processor or ac cording to the formula (门，the blank value should be subtracted from the above result of calcula tion. 12 SN/T 1973-2007 X=A:c , V -一As.m ) 1 ( . . . . . . . . . . . . . . . . . . . . . . . . . . where X -the content of abame
44、ctin in the test sample. mg/kg; c一theconcentration of abamectin in the standard working solution.闯/mL;V-the final volumn of the sample solution. mL; A -the peak area of abamectin in the sample solution; As一thepeak area of abamectin in the standard working solution; m-the corresponding mass of test s
45、ample in the final solution. g. B 川叫8. 1川limit内m时刷n时巾1甘山i忧t。刷f忖d阳minatJ ./ /,J 8. 2 Recovery Fortifying concentrations i test samples and recovery expermental data are list in table 2. Table 2-f!.or1ifying.coocentr.ations .and.recOiery experimental data Sample /F石巾市协CEment酬。n!(mg/kgl/伽5/ 0.01 O. 05
46、nce I 67.2o/d -99 (0/0 74.6%亏90.8%70.0% -86. 6% garlic 61.6%1-85 % .2%.! 83.4% 72. 3% -84. 9% spinach 68.P%-1O 8% 争8%才99.6%81.7%-9日.1%apple 69机-H/J.8%I 卢6%076%73.1%-91.7% chestnut 17B4%)=1112%Jf 4 ( 86.4%千102.6%72.6%-83.6% tea X2%.94.0% 74，8%儿85.8%69.6% -90. 6% beef 、s%-95. o/o 74_.6%-92.2% 74.1%-99
47、.1% 飞Jn、utton66.0% -76.8% 74.8% -88. 4% 68.3%-79.7% chicken 64.4% -85.6% 74.4% .96. 0% B口.-4%-101.3%fish 73.2% -96. 8% 74.6% -103. 6% 85.2% -97. 2% honey 74.0% -97. 2% 74.8%-102.2% 66.6%-87.3% chishao 64.8% -85. 2% 74.6% -87. 0% 70.9% -96. 9% vlnegar 67.2%-74.4% 65.6% -87. 8% 76.2%-85.3% 13 SN/T 197
48、3-2007 A.l Nebulizer Pressure: 60 psi. A. 2 Drying Gas Flow: 5 L/min. A. 3 Drying Gas Temp: 350C. A. 4 APCI Vap Temp: 400C. A. 5 Corona Current: 10 000 nA. A.6 Capillary Voltage: 3500 V. Annex A (informative annex) Mass spectrometry parameters 1) Non-commericial statement 1 the equipments and their
49、ty阳sAgilent LC-MSD-Tra p-VL involved in the standard method are not related to commercial aims. and the analysts are encouraged to use equipents of dfferent cor poration or different type 14 Annex B (informative annex) SN/T 1973-2007 Selected ion chromatograms of abamectin standard and daughter ion mass spe
