1、版权所有 禁止翻制、电子传阅、发售中华人民共和国出入境检验检疫行业标准SN/T 1921-2007 进出口动物源性食品中氟甲睡残留量检测方法液相色谱-质谱/质谱法Determination of residue of flumequine in foodstuffs of animal origin for import and export-LC-MS/MS method 2007-05-23发布2007-12-01实施中华人民共和国发布国家质量监督检验检度总局版权所有 禁止翻制、电子传阅、发售目。吕本标准附录A和附录B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。本标准由中国
2、检验检疫科学研究院负责起草。本标准主要起草人:彭涛、安娟、国伟、李晓娟、严矛、徐历、唐英章、储晓刚。本标准系首次发布的出入境检验检疫行业标准。SN/T 1921-2007 版权所有 禁止翻制、电子传阅、发售1 范围进出口动物源性食品中氟甲睡残留量检测方法液相色谱-质谱/质谱法本标准规定了动物源性食品中氟甲喳残留量液相色谱质谱/质谱测定方法。本标准适用于动物组织、内脏、蛋、奶、鱼和虾中氟甲喳残留量的检测。2 试样制备与保存2.1 动物组织、内脏、鱼和虾SN/T 1921-2007 从原始样品取出有代表性样品约500g，用组织捣碎机充分捣碎?昆匀，均分成两份，分别装入洁净容器作为试样，密封，并标明
3、标记。将试样置于18C冷冻避光保存。2.2蛋从原始样品取出有代表性样品约500g，去壳后用组织捣碎机搅拌充分泪匀，均分成两份，分别装入洁净容器作为i式样，密封，并标明标记。将同样置于4c冷藏避光保存。2.3奶从原始样品取出有代表性样品约500g，用组织捣碎机充分由匀，均分成两份，分别装入洁净容器作为试样，密封，并标明标记。将试样置于4C冷藏避光保存。在制样的操作过程中，应防止样品污染或发生残留物含量的变化。3 方法提要用磷酸盐缓冲液和乙睛提取试样中残留的氟甲哇，经正己院去除脂肪、C18固相萃取柱净化后，采用液相色谱质谱/质谱检测，外标法定量。4 试剂和材料除非另有说明，所有试剂均为分析纯，水为
4、去离子水或相当纯度的水。4.1 乙睛:高效液相色谱级。4.2 正己皖:高效液相色谱级。4.3 甲醇:高效液相色谱级。4.4 磷酸。4.5 磷酸二氢饵。4.6 氢氧化纳。4. 7 氨水:25%。4.8 甲酸:高效液相色谱级。4.9 乙酸钱。4.10 乙睛饱和的正己皖:向100mL乙睛中加入100mL正己院，充分振荡后，静置分层，取上层乙睛备用。4.11 磷酸盐缓冲液(0.05mol/L , pH7. 4士0.2):准确移取250mL O. 2 mol/L磷酸二氢何水溶液和197. 5 mL O. 2mol/L氢氧化纳水溶液于1L容量瓶中，再加水500mL，用磷酸或氢氧化纳调节pH7.4士0.2，
5、用水定容至1L。版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 4.12 SPE洗脱液:向75mL甲醇中加入25mL氨水，由匀备用。4.130.1%甲酸水溶液(含0.5mmol/L乙酸锻):准确量取1mL甲酸和称取o.0386 g乙酸锻于1L 容量瓶中，用水定容至1L4.14 标准品:氟甲哇，纯度二三99%。4.15 标准储备液:准确称取适量氟甲喳标准品(精确至o.000 1 g)，用乙睛溶解，配制成浓度为100 mg/L的标准储备溶液，-180C冷冻避光保存3个月。4.16 中间标准溶液:准确移取1mL标准储备液于10mL容量瓶中，用乙睛定容至刻度，配制成浓度为10mg/L的
6、中间标准溶液，40C冷藏避光保存1个月。4.17 标准工作溶液:根据需要用0.1%甲酸水溶液(4.13)稀释成适用浓度的标准工作溶液，现用现配。4.18 Cl8固相萃取柱:3mL, 500 mg，或相当者。4.19 微孔滤膜:0.2m，有机相。4.20 氮气:纯度二三99.999%。5 仪器和设备5.1 液相色谱质谱/质谱仪:配备电喷雾离子源(ESD。5.2 组织捣碎机。5.3 均质器:10 000 r/mino 5.4 振荡器。5.5 离心机:10 000 r/mino 5.6 氮吹仪。5. 7 涡动仪。5.8 超声波水浴。5.9 容量瓶:1 L , 10 mL。5.10 塑料离心管:50m
7、Lo 5.11 分液漏斗:125 mLo 5.12 量筒:50mLo 5.13 刻度试管:10mLo 6 测定步骤6.1 提取6.1.1 动物组织、内脏、鱼和虾称取约2g试样(精确至0.01g)于50mL塑料离心管中，加入18mL磷酸盐缓冲液(4.11)和2mL 乙睛，用均质器以10000 r/min均质2min后，再以10000 r/min离心10mino收集上清液于125mL 分液漏斗中，加入20mL乙腊饱和后的正己皖(4.10)，振荡10min后，静置20mi口，收集全部下层液体于50mL量筒中，用水定容至30mL 6.1.2 蛋和奶称取约2g试样(精确至0.01g)于50mL塑料离心管
8、中，加入18mL磷酸盐缓冲液(4.11)和2mL 乙睛，用振荡器振摇提取20min后，再以10000 r/min离心20min。余下操作同6.1.106.2 净化C18固相萃取柱(4.18)依次用3mL甲醇、6mL水预淋洗后，准确转入15mL样品提取液(6.1)。用3mL水进行淋洗，弃去;再用5mL SPE洗脱液(4.12)进行洗脱，收集洗脱液于10mL刻度试管中。整个固相萃取净化过程控制流速不超过2mL/mi口。洗脱液在400C下用氮气吹干。残留物用1mL 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 0.1%甲酸水榕液(4.13)溶解，涡旋1min后，过0.2m微孔滤膜(
9、4.19)，供仪器测定。6.3 测定6.3. 1 液相色谱条件6.3. 1. 1 色谱柱:C18, 150 mmX2. 1 mm(内径)，5m，或相当者。6.3. 1. 2 流动相:乙腊十0.1%甲酸水溶液(45十55，体积比)。6.3. 1. 3 柱温:300C。6.3. 1. 4 流速:0.2mL/mino 6.3. 1. 5 进样量:10L6.3.2 质谱条件参见附录Ao6.3.3 液相色谱质谱/)贡谱测定根据样液中氟甲喳的含量，选定浓度相近的标准工作榕液，待测样液中氟甲喳的响应值应在仪器检测的线性范围内。对标准工作溶液及样液(6.2)等体积参插进样测定。在上述仪器条件下，氟甲喳保留时间
10、约为4.1mino液相色谱质谱/质谱色谱图参见附录B图B.L6.3.4 阳性样品的确证按照上述条件测定样品和标准工作液，如果检测的质量色谱峰保留时间与标准工作液一致，允许偏差小于士2.5%;定性离子对的相对丰度与浓度相当标准工作液的相对丰度一致，相对丰度偏差不超过表1的规定，则可判断样品中存在相应的被测物。表1定性测定时相对离子丰度的最大允许偏差相对离子丰度/c%)允许的相对偏差/C%)6.4 空白试验50 士2020至50士25除不加试样外，按上述测定步骤进行。7 结果计算和表达试样中的残留含量，按式(1)计算:式中:x= AXcXV -A , X rn X 1 000 X 试样中氟甲喳残留
11、含量，单位为毫克每千克(mg/kg); A 样液中氟甲喳的峰面积;A 标准工作溶液中氟甲喳的峰面积;10至20士30c 标准工作榕液中氟甲喳浓度，单位为纳克每毫升(ng/mL); V 样液最终定容体积，单位为毫升(mL); m 样液所代表最终试样的质量，单位为克(g)。注:计算结果需将空白值扣除。8 测定低限和回收率8.1 测定低限本标准的测定低限为:0.0005mg/kgo 8.2 回收率鸡肉:添加浓度O.000 5 mg/kg，回收率为70.0%106. 0%; 添加浓度0.005mg/kg，回收率为76.0%106. 0%; :S:; 10 士50. ( 1 ) 3 版权所有 禁止翻制、
12、电子传阅、发售SN/T 1921-2007 4 添加浓度0.05mg/kg，回收率为80.4%100. 0%。鸡蛋:添加浓度0.0005 mg/kg，回收率为72.0%106. 0%; 添加浓度0.005mg/kg，回收率为82.0%104. 0%; 添加浓度0.05mg/kg，回收率为81.6%96. 2%。猪肝:添加浓度O.000 5 mg/kg，回收率为76.0%106. 0%; 添加浓度0.005mg/kg，回收率为82.0%98. 0%; 添加浓度0.05mg/kg，回收率为84.0%95. 8%。牛奶:添加浓度0.0005 mg/kg，回收率为76.0%104. 0%; 添加浓度0
13、.005mg/kg，回收率为74.0%96. 0%; 添加浓度0.05mg/kg，回收率为86.8%98. 4%。鱼:添加浓度O.000 5 mg/kg，回收率为78.0%106. 0%; 添加浓度0.005mg/kg，回收率为82.0%104. 0%; 添加浓度0.05mg/kg，回收率为84.2%102. 4%。虾:添加浓度0.0005 mg/kg，回收率为76.0%106. 0%; 添加浓度0.005mg/kg，回收率为72.0%96. 0%; 添加浓度0.05mg/kg，回收率为81.8%95. 2%。版权所有 禁止翻制、电子传阅、发售电离方式毛细管电压源温度去溶剂温度锥孔气流去溶剂气
14、流碰撞气压监测模式化合物母离子氟甲噎262 a 离子用于定量。附录Al)(资料性附录)质谱条件表A.1质谱条件表A.2多反应监测条件子离子驻留时间/s244 O. 20 202 O. 20 SN/T 1921-2007 ESI十3.0 kV 120C 350C 氮气.100L/h 氮气.600L/h 氧气.2.40 X 10 6 la 多反应监测锥孔电压/V碰撞能量/cV25 17 25 31 1)附录A所列参数是在WatcrsQuattro lrcmicr质谱仪上完成的，此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 版权所有 禁止翻
15、制、电子传阅、发售SN/T 1921-2007 100 % 。100 % 。6 1. 00 2.00 附录B(资料性附录)标准物质色谱固3.00 4.00 4.1 min 262244 262202 岳006. 00 图B.1氟甲睡液相色谱质谱/质谱多反应监测色谱图limc 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 Foreword Annex A and Annex B of this standard are informative annexes. This standard was proposed by and is under the jurisdiction
16、 of the Certification and Accreditation Administration of the Peoples Republic of China. This standard is drafted by Chinese Academof Inspection and Quarantine. Main drafters of this standard are: Peng Tao , An Juan , Guo Wei , Li Xiaojuan , Yan Mao , Xu Li , Tang Yingzhang and Chu Xiaogang. This st
17、andard is a professional standard for Entry-Exit inspection and quarantine of the Peoples Re public of China promulgated for the first time. Note: This English Version. a translation from the Chinese text. is solely for guidance. 7 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 1 Scope Determination of residue of
18、flumequine in foodstuffs of animal origin for import and export-LC-MS/MS method This standard specifies the determination of flumequine residue in animal origin foodstuffs for import and export by LC-MS/MS. This standard is applicable to the determination of flumequine residue in animal tissue , off
19、all , egg , milk, fish , and shrimp. 2 Sample preparation and storage 2. 1 Animal tissue, offall , fish and shrimp AII primary sample is reduced to 500 9 as the representative sample , which is blended and homoge nized , and then divided into two equal portions. Each portion is placed in clean conta
20、iners as the test sample , which is sealed and labeled. The test sample should be stored below in -lSoC avoiding sun light. 2.2 Egg AII primary sample is reduced to 500 9 as the representative sample , which is shelled and homoge nized , and then divided into two equal portions. Each portion is plac
21、ed in clean containers as the test sample , which is sealed and labeled. The test sample should be stored below in 40C avoiding sun light. 2.3 Milk AII primary sample is reduced to 500 9 as the representative sample , which is homogenized , and then divided into two equal portions. Each portion is p
22、laced in clean containers as the test sample , which is sealed and labeled. The test sample should be stored below in 40C avoiding sunlight. In the course of sample preparation , precautions should be taken to avoid contamination or anfactors which may cause the change of residue content. 8 版权所有 禁止翻
23、制、电子传阅、发售SN/T 1921-2007 3 Principle The flumequine residue in the test sample is extracted with phosphate buffer and acetonitrile. After cleaned up with n-hexane and C18 SPE cartridge, the residue is then determined by LC-MS/MS, and quantified by external standard method. 4 Reagents and materials Un
24、less otherwise specified , all regents used are A. R. , and pure water is redistilled water. 4.1 Acetonitrile: HPLC grade. 4.2 N-hexane: HPLC grade. 4.3 Methanol: HPLC grade. 4.4 Phosphoric acid. 4.5 Potassium dihydrogen phosphate. 4.6 Sodium hydroxide. 4.7 Ammonia: 25%. 4.8 Formic acid: HPLC grade.
25、 4.9 Ammonium acetate. 4.10 N-hexane saturated with acetonitrile: add 100 mL n-hexane into 100 mL acetonitrile, mix ad-equately, then wait for delamination , use the substrate layer. 4.11 Phosphate buffer (0.05 mol/L pH7. 4 :t 0.2): add 250 mL 0.2 mol/L potassium dihydrogen phosphate solution , 197.
26、5 mL O. 2 mol/L sodium hydroxide solution and 500 mL water into 1 L Volu metric flask , then adjust pH7. 4 :t 0. 2 with phosphoric acid or sodium hydroxide solution , diluted to 1 L with water. 4.12 SPE eluent solution: add 25 mL 25% ammonia into 75 mL methanol , mix adequatel. 4.13 O. 1 % formic ac
27、id solution (containing 0.5 mmol/L ammonium acetate): add 1 mL formic acid and 0.038 6 9 ammonium acetate into 1 L volumetric flask , then dilute to 1 L with water. 4.14 Flumequine standard: Purity二三99%.9 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 4. 15 Standard stock solution: accurately weigh an adequate amo
28、unt of flumequine standard , dis solve in acetonitrile and prepare a solution of 100 mg/L as the standard stock solution , stored below -1SC avoiding sunlight for three months. 4. 16 Middle standard solution: accurately transfer 1 mL standard stock solution into 10 mL volu metric flask, then make up
29、 to graduation with acetonitrile, prepare a solution of 10 mg/L as the mid dle standard solution , stored below 4巳avoidingsunlight for a month. 4.17 Standard working solution: according to the requirement , dilute middle standard solution to appropriate concentration with O. 1 % formic acid solution
30、 (4. 13)just before use. 4.18 CB SPE cartridge: 3 mL, 500 mg , or equivalent. 4.19 Membrane filter: 0.2m， organic type. 4.20 Ntrogen: purity二三99.999%.5 Apparatus and equipment 5.1 Liquid chromatography-tandem mass spectrometr， equipped with electrospray ion source. 5.2 Tissue blender. 5.3 Homogenize
31、r: 10000 r/min. 5.4 Shaker. 5.5 Centrifuge: 10000 r/min. 5.6 Nitrogen evaporator. 5. 7 Vortex mixer. 5.8 Ultrasonic water bath. 5.9 Volumetric flask: 1 L, 10 mL. 5.10 Plastic centrifuge tube: 50 mL. 5.11 Separatingfunnel: 125mL. 5.12 Volumetric cylinder: 50 mL. 10 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 5.1
32、3 Graduated cuvette: 10 m L. 6 Procedure 6. 1 Extraction 6.1.1 Animal tissue, offall, fish and shrimp Weigh about 2 9 (accurate to O. 01g) test sample into a 50 mL plastic centrifuge tube, then add 18 mL phosphate buffer (4. 11) and 2 mL acetonitrile into the centrifuge tube. Homogenize at 10000 r/m
33、in for 2 min. before centrifuge at 10000 r/min for 10 min , Transfer the supernatant into a 125 mL separating funnel , then add 20 mL n-hexane saturated with acetonitrile (4.10). Shake for 10 min and then wait for 20 min. Collect the substrate laer to a 50 mL volumetric cylinder, and make up to 30 m
34、L with water. 6.1.2 Egg and milk Weigh about 2 9 (accurate to 0.01 g) test sample into a 50 mL plastic centrifuge tube, then add 18 mL phosphate buffer (4.11 )and 2 mL acetonitrile into the centrifuge tube. Shake for 20 min , be fore centrifuge at 10000 r/min for 20 min. The remaining procedure is t
35、he same as 6. 1. 1. 6.2 Clean up The C18 SPE cartridge (4.18) is conditioned with 3 mL methanol and 6 mL water in sequence. After 15 mL extract solution (6. 1) is loaded , wash the cartridge with 3 mL water, and discard the efflu ent. Elute with 5 mL SPE eluent solution (4.12) , and collect all the
36、eluate into a 10 mL graduated cu vette. Adjust the flow rate below 2 mL/min during all the SPE process. Evaporate the eluate to dry ness under nitrogen at 400C. The residue is reconstituted with 1 mL O. 1 % formic acid solution (4.13) , then vortex for 1 min and filtrate through a membrane filter (4
37、. 19). The filtrate is ready for LC-MS/MS determination. 6.3 Determination 6.3. 1 HPLC operating condition 6.3.1.1 LC column: C18, 150 X 2.1 mm (i巾，5m， or equivalent. 6.3.1.2 Mobile phase: acetonitrile +0.1% formic acid (45+55 , V/V). 6.3.1.3 Column temperature: 300C. 6.3.1.4 Flow rate: 0.2 mL/min.
38、11 版权所有 禁止翻制、电子传阅、发售SN/T 1921-2007 6.3. 1. 5 Injection volume: 10L. 6.3. 2 MS/MS operating condition See annex A. 6.3.3 LC-MS/MS determination According to the estimated approximate concentration of flumequine in the sample solution , select the standard working solution of similar concentration to
39、that of sample solution. The responses of flumequine in the sample solution should be in the linear range of the instrumental detection. The standard working solution should be injected randomlin-between the injections of the sample solu tion (6. 2) of equal volume. Under the above instrumental cond
40、ition , the retention time of flume quine is ca. 4.1 min. For the chromatogram of flumequine standard , see annex B. Fig B. 1. 6.3.4 Confirmation Under above determination condition , the variation range of the retention time for the peak of ana Iyte in unknown sample and in the standard working sol
41、ution can not be out of range of :t 0.25 min. For the same analysis batch and the same compound , the variation range of the ion ratio between the two daughter ions for the unknown sample and the standard working solution at the similar concen tration can not be out of range of table 1 , and then th
42、e corresponding analte must be present in the sample. Table 1-Maximum permitted tolerances for relative ion intensities while confirmation Relative intensity 50% 20% to 50% 10% to 20% 10% Permitted tolerances :t 20% :t 25% :t 30% :t 50% 6.4 Blank te5t The operation of the blank test is the same as t
43、hat described in the method of determination , but with omission of sample addition. 7 Calculation and expression of result Calculate the residue content of flumequine in the test samples according to the formula (1) : AxcxV As x m x 1 000 where X-the residue content of flumequine in the test sample
44、, mg/kg; A-the peak area of flumequine in the test sample solution; As-the peak area of flumequine in the standard working solution; 12 . ( 1 ) 版权所有 禁止翻制、电子传阅、发售c-the concentration of flumequine in the standard working solution , ng/mL; V-the final volume of sample solution , mL; m-the corresponding
45、 mass of test sample in the final sample solution , g. Note: The blank value should be subtracted from the result of calculation. 8 Limit of determination and recovery 8. 1 Li mit of determination The limit of determination of this method is 0.000 5 mg/kg. 8. 2 Recovery For the chicken , data of the
46、 spiking level and the recovery are: When the spiking level is 0.000 5 mg/kg , the recovery is 70.0% - 1 06.0% ; When the spiking level is 0.005 mg/I甸，the recovery is 76.0 % - 1 06.0% ; When the spiking level is 0.05 mg/闸，the recovery is 80.4%-100.0%. For the egg , data of the spiking level and the
47、recovery are: When the spiking level is 0.000 5 mg/kg , the recoveris 72.0% -106.0%; When the spiking level is 0.005 mg/闸，the recovery is 82.0 % - 1 04.0% ; When the spiking level is 0.05 mg/I甸，the recovery is 81.6% -96.2%. For the liver, data of the spiking level and the recoverare: When the spikin
48、g level is 0.000 5 mg/kg , the recovery is 76.0% - 1 06.0% ; When the spiking level is 0.005 mg/I甸，the recovery is 82.0 % -98.0% ; When the spiking level is 0.05 mg/闸，the recovery is 84.0%-95.8%. For the milk, data of the spiking level and the recovery are: When the spiking level is 0.000 5 mg/kg ,
49、the recoveris 76.0% -104.0%; When the spiking level is 0.005 mg/闸，the recovery is 74.0% -96.0%; When the spiking level is 0.05 mg/I甸，the recovery is 86.8%-98.4%. For the fish , data of the spi king level and the recovery are: When the spiking level is 0.000 5 mg/kg , the recovery is 78.0% - 1 06.0% ; When the spiking level is 0.005 mg/I甸，the recovery is 82.0 % - 1 04.0% ; When the spiking level is 0.05 mg/闸，the recovery is 84.2%-102.4
