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 1922-2007 进出口动物源性食品中对乙酷氨基盼、邻乙田先水杨酸残留量检测方法液相色谱-质谱/质谱法Determination of residues of paracetamol and acetylsalicylic acid in foodstuffs of animal origin for import & 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疫总局版权所有 禁止翻制、电子传阅、发售中华人民共和国出入境检验检
2、疫行业标准进出口动物源性食品中对乙酷氨基酣、邻乙酷水杨酸残留量检测方法夜相色i普-质i普/贡谱法S:f/T 1922-2007 关中国标准出版社出版北京复兴门外三里河北街16号邮政编码:100045网址www.spc.口et.c口电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷关开本880X1230 1/16 印张1.25 字数33千字2007年9月第一版2007年9月第一次印刷印数1-2000今令书号:155066 2-18065 定价12.00兀版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 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
3、监督管理委员会提出并归口。本标准由中国检验检疫科学研究院、中华人民共和国深圳出入境检验检疫局负责起草。本标准主要起草人:彭涛、于静、李晓娟、岳振峰、李建中、国伟、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。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对乙酷氨基酣、邻乙酷水杨酸残留量检测方法液相色谱-质谱/质谱法SN/T 1922-2007 本标准规定了动物源性食品中对乙眈氨基酷、邻乙酷水杨酸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、蛋和奶中对乙酷氨基酷、邻乙酷水杨酸残留量的检测。2 试样制备与保存2.1 动物组织和内脏从原始样品取出有代表性样
4、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混匀，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，用组织捣碎机充分混匀，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 方法提要用乙睛提取试样中残留的对乙眈氨基酷、邻乙酷水杨酸(残留标示物:水杨酸)，经正己院去除脂
5、肪、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 甲酸:高效液相色谱级。4.6 乙酸绩。4. 7 乙腊饱和的正己皖:向100mL乙睛中加入100mL正己:院，充分振荡后，静置分层，取上层乙腊备用。4.8 o. 1 %甲酸水溶液(含0.5mmol/L乙酸锻):准确量取1mL甲酸和称取O.038 6 g乙酸镀于1L 容量瓶中，用水定容至1L 4.9 SPE淋洗液:0.02mol/L磷酸+甲醇(80十
6、20，体积比)。4.10 标准品:对乙眈氨基酌，纯度二三99%;水杨酸，纯度二三99%。4.11 标准储备溶液:称取对乙眈氨基酷、水杨酸的标准品(精确至0.0001 g)，用乙腊溶解，配制成浓版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 度为100mg/L的标准储备、溶液，-180C冷冻避光保存3个月。4.12 中间泪合标准溶液:分别移取1mL单个药物的标准储备液于10mL容量瓶中，用乙腊定容至刻度，配制成浓度为10mg/L的中间混合标准溶液，40C冷藏避光保存1个月。4.13 混合标准工作溶液:根据需要用0.1%甲酸水、溶液(4.8)稀释中间由合标准榕液(4.12)成适合浓
7、度的混合标准工作溶液，现用现配。4. 14 Oasis HLB固相萃取柱:6mL, 200 mg，或相当者。4.15 微孔滤膜:0.2m，有机相。4.16 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(ESl)。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 减压浓缩仪。5. 7 氮吹仪。5.8 涡动仪。5.9 超声波水浴。5.10 容量瓶:1L , 10 mLo 5.11 塑料离心管:50mLo 5.12 分液漏斗:125mL 5.13 刻度试管:10mL 6 测
8、定步骤6.1 提取6.1.1 动物组织和内脏称取约2g试样(精确至O.01 g)于50mL塑料离心管中，加入20mL乙脯，用均质器以10 000 r/min均质2min后，再以10000 r/mi口离心10mi口，收集上清液于125mL分液漏斗中。残渣用20mL乙睛再提取一次，合并上清液。加入20mL乙睛饱和后的正己皖(4.7)，振荡10min后，静置20 min，收集下层液体。在400C水浴中减压浓缩至干，残留物用5mL SPE淋洗液(4.9)溶解，超声波水浴助溶5min后，待净化。6.1.2 蛋和奶称取约2g试样(精确至0.01g)于50mL塑料离心管中，加入20mL乙睛，用振荡器振摇提取
9、20 min后，再以10000 r/mi口离心10mi口，余下操作同6.1.106.2 净化Oasis HLB固相萃取柱(4.14)依次用5mL甲醇、5mL水、5mL O. 02 mol/L磷酸预淋洗后，转入5 mL样品提取液(6.1)。先用5mL SPE淋洗液(4.9)进行淋洗，弃去;再用5mL水进行淋洗，弃去;最后用5mL甲醇进行洗脱，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2 mL/mi口。?先脱液在400C下用氮气吹干。残留物用1mL O. 1%甲酸水溶液(4.8)溶解，涡旋1min 后，过0.2m微孔滤膜，供仪器检测。版权所有 禁止翻制、电子传阅、发售6.3
10、 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:A:乙睛，B:0.1%甲酸水榕液(4.的。6.3. 1. 3 梯度洗脱:见表L监测时间/min。114 49 916 6.3. 1. 4 柱温:30 oC 0 6.3. 1. 5 流速:0.2mL/mino 6.3. 1. 6 进样量:20Lo6.3.2 质谱条件参见附录Ao6.3.3 液相色谱-质谱/贡谱测定表1梯度洗脱条件流动相A/C%)10 1090 90 10 SN/T 1922-2007 流动相B/C%)90 9010 10 90 根
11、据样液中待测物的含量，选定浓度相近的标准工作榕液，待测样液中对乙眈氨基酷、水杨酸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，对乙眈氨基酷、水杨酸保留时间分别约为5.5min、9.0mino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2的规定，则可判断样品中存在相应的被测物。表2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
12、许的相对偏差/C%)6.4 空白试验50 士20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:20至50士25X AXcXV A , X rn X 1 000 式中:X 试样中药物残留含量，单位为毫克每千克(mg/kg); A 样液中药物的峰面积;人标准工作榕液中药物的峰面积;c 标准工作溶液中药物浓度，单位为纳克每毫升(口g/mL); V 样液最终定容体积，单位为毫升(mL);10至20士30三二10士50. ( 1 ) 3 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 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
13、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:对乙眈氨基酣o.000 5 mg/kg;邻乙眈水杨酸(以水杨酸计)0.01mg/kg o 8.2 回收率8.2. 1 对乙酷氨基酣猪肉:添加浓度o.000 5 mg/kg，回收率为86.0%110. 0%; 添加浓度0.05mg/kg，回收率为80.6%98. 4%; 添加浓度0.1mg/kg，回收率为81.4%94. 4%。猪肝:添加浓度o.000 5 mg/kg，回收率为84.0%1l6. 0%; 添加浓度0.05mg/kg，回收率为80.6%102. 8%; 添加浓度0.1mg/阳，回收率为81.2%95. 8%。鸡蛋:添
14、加浓度O.000 5 mg/kg，回收率为72.0%102. 0%; 添加浓度0.05mg/kg，回收率为82.0%96. 6%; 添加浓度0.1mg/kg，回收率为80.3%91. 6%。牛奶:添加浓度O.000 5 mg/kg，回收率为66.0%90. 0%; 添加浓度0.05mg/kg，回收率为81.2%97. 4%; 添加浓度0.1mg/阳，回收率为80.3%93. 6%0 8.2.2 邻乙酷水杨酸(以水杨酸计)4 猪肉:添加浓度0.01mg/kg，回收率为82.0%104. 0%; 添加浓度0.05mg/kg，回收率为81.6%101. 6%; 添加浓度0.2mg/阳，回收率为80.
15、6%95. 9%。猪肝:添加浓度0.01mg/kg，回收率为80.0%107. 0%; 添加浓度0.05mg/kg，回收率为80.4%105. 8%; 添加浓度0.2mg/kg，回收率为83.5%91. 1%。鸡蛋:添加浓度0.01mg/kg，回收率为82.0%104. 0%; 添加浓度0.05mg/kg，回收率为80.8%99. 0%; 添加浓度0.2mg/阳，回收率为81.2%95. 9%。牛奶:添加浓度0.01mg/kg，回收率为86.0%109. 0%; 添加浓度0.05mg/kg，回收率为80.4%97. 8%; 添加浓度0.2mg/kg，回收率为80.2%94. 9%。版权所有 禁
16、止翻制、电子传阅、发售质谱参数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流碰撞气压监测模式化合物母离子对乙酷氨基盼152 水杨酸137 a 离子用于定量。附录Al)(资料性附录)质谱条件表A.1质谱条件对乙酷氨基盼ESH 3.0 kV 1200C 3500C 氮气.100L/h 氮气.600L/h 氧气.2.40X106Pa 多反应监测表A.2多反应监测条件SN/T 1922-2007 水杨酸ESI 子离子驻留日才间/s锥孔电压/V碰撞能量/eV93 0.2 s 25 V 22 eV 110日0.2 s 25 V 16 eV 65 0.2 s 30 V 26 cV 93 0.2 s 3
17、0 V 15 cV 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 禁止翻制、电子传阅、发售SN/T 1922-2007 附录B (资料性附录)标准物质色谱固100 5.5 min 1岳2110, , % 。100 1岳293% 。lme 2岳。岳.007.岳。10.00 12.岳。I岳.00100 9.0 mm 13793 % 。100 13765 % 。lme 2.50 5.00 7.50 10.00 12.50 15.00 (按保留时间依次为
18、2对乙眈氨基盼和水杨酸)固B.1液中目色i晋-质i晋/质谱多反应监测色i晋图6 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 Foreword Annex A and Annex B of this standard are informative annexes. This standard was proposed by and is under the jurisdiction of the Certification and Accreditation Administration of the Peoples Republic of China. This stan
19、dard is drafted by Chinese Academy of Inspection and Ouarantine and Shenzhen Entry-Exit Inspection and Ouarantine Bureau of PRC. Main drafters of this standard are:Peng Tao , Yu Jing ,Li Xiaojuan , Yue Zhengfeng , Li Jianzhong ,Guo Wei , Tang Yingzhang and Chu Xiaogang. This standard is a profession
20、al Standard for entry-exit inspection and quarantine 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 1922-2007 Determination of residues of paracetamol and acetylsalicylic acid in foodstuffs of animal
21、origin for import & export -LC-MSjMS method 1 Scope This standard specifies the determination of paracetamol and acetylsalicylic acid residues in animal origin foodstuffs by LC-MS/MS. This standard is applicable to the determination of paracetamol and acetylsalicylic acid residues in an imal tissue
22、, offal , egg and milk. 2 Sample preparation and storage 2. 1 Animal tissue and offall 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 containers as the test sample , w
23、hich 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 placed in clean containers as the
24、 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 placed in clean containers as
25、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 any fac tors which macause the change of residue content. 8 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 3 Pr
26、inciple The paracetamol and acetIsaliclic acid Cresidue marker: salicylic acid) residues in the test sample are extracted with acetonitrile. After cleaned up with n-hexane and HLB SPE cartridge, the residues are then determined bLC-MS/MS， and quantified by external standard method. 4 Reagents and ma
27、terials Unless 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 Formic acid: HPLC grade. 4.6 Ammonium acetate. 4.7 N-hexane saturated with acetonitrile: a
28、dd 100 mL n-hexane into 100mL acetonitrile, mix ade-quatel，then wait for delamination , use the substrate layer. 4.8 0.1% formic acid solution Ccontaining 0.5 mmol/L ammonium acetate): add 1 mL formic acid and 0.0386 9 ammonium acetate into 1 L volumetric flask, then dilute to 1 L with water. 4.9 SP
29、E syringe solution: 0.02 mol/L phosphoric acid+methanol (80+20, V/V). 4.10 Paracetamol and salicylic acid standard: purity二三99%.4. 11 Standard stock solution: accurately weigh an adequate amount of paracetamol and salicylic acid standards ,dissolve in acetonitrile and prepare a solution of 100 mg/L
30、as the standard stock solu tion , stored below -18巳avoidingsunlight for three months. 4.12 Middle mix standard solution: accurately transfer 1 mL each drug standard stock solution into the same 10 mL volumetric flask respectively, then make up to graduation with acetonitrile, prepare a solution of 1
31、0 mg/L as the middle mix standard solution , stored below 40C avoiding sunlight for a month. 9 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 4.13 Mix standard working solution: according to the requirement , dilute middle mix standard so lution to appropriate concentration with O. 1 % formic acid solution (4.8) j
32、ust before use. 4.14 Oasis HLB SPE cartradge: 6 mL, 200 mg , or equivalent. 4.15 Membrane filter: 0.2m， organic type. 4.16 Nitrogen: purit二三99.999%.5 Apparatus and equipment 5.1 Liquid chromatography-tandem mass spectrometry, equipped with electrospray ion source. 5.2 Tissue blender. 5.3 Homogenizer
33、: 10000 r/min. 5.4 Shaker. 5.5 Centrifuge: 10000 r/min. 5.6 Rotary vacuum evaporator. 5. 7 Nitrogen evaporator. 5.8 Vortex mixer. 5.9 Ultrasonic water bath. 5.10 Volumetric flask: 1 L, 10 mL. 5.11 Plastic centrifuge tube: 50 mL. 5.12 Separating funnel: 125 mL. 5.13 Graduated cuvette: 10 m L. 6 Proce
34、dure 6. 1 Extraction 10 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 6. 1. 1 Animal tissue and offall Weigh about 2 9 (accurate to 0.01 g) test sample into a 50 mL plastic centrifuge tube, then add 20 mL acetonitrile into the centrifuge tube. Homogenize at 10000 r/min for 2 min , before centrifuge at 10000 r/min
35、 for 10 min , Transfer the supernatant into a 125 mL separating funnel. The residue is extracted again with another 20 mL acetonitrile. And combine the supernatant into the same separa ting funnel. Then add 20 mL n-hexane saturated with acetonitrile (4.7). Shake for 10 min and then wait for 20 min.
36、Collect the substrate layer and evaporate to drness with rotarevaporator in a wa ter-bath at 40C. The residue is then reconstituted with 5 mL SPE syringe solution (4.9). After dis solved assisted with ultrasonic water-bath for 5 min, the solution is ready for clean up. 6.1.2 Egg and milk Weigh about
37、 2 9 (accurate to O. 01 g) test sample into a 50 mL plastic centrifuge tube , then add 2 mL acetonitrile into the centrifuge tube. Shake for 20 min , before centrifuge at 10000 r/min for 20 min. The remaining procedure is the same as 6. 1. 1. 6.2 Clean up The Oasis HLB cartridge (4. 14) is condition
38、ed with 5 mL methanol , 5 mL water and 5 mL O. 02 mol/L phosphoric acid in sequence. After the extract solution (6. 1) is loaded , wash the cartridge with 5 mL SPE sringe solution(4. 9) and 5 mL water, discard the effluent. Then elute with 5 mL metha nol , collect all the eluate into a 10 mL graduat
39、ed cuvette. Adjust the flow rate below 2 mL/min dur ing all the SPE process. Evaporate the eluate to dryness under nitrogen at 40C. The residue is re constituted with 1 mL O. 1 % formic acid solution (4.酌，then vortex for 1 min and filtrate through a membrane filter (4.15). The filtrate is ready for
40、LC-MS/MS determination. 6. 3 Determination 6.3. 1 HPLC operating condition 6.3.1.1 HPLCcolumn: C8 150X2.1 mm (id) , 5m， or equivalent. 6.3.1.2 Mobile phase: A: acetonitrile，也O.1 % formic acid solution (4.8). 6.3. 1.3 Gradient elution: see table 1. Table 1-Gradient elution condition Time window/min M
41、obile phase A/ (% ) Mobile phase B/(%) 0-1 10 90 1-4 10-90 90-10 4-9 90 10 9-16 10 90 11 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 6.3.1.4 Column temperature: 300C. 6.3.1.5 Flow rate: 0.2 mL/min. 6.3. 1. 6 Injectoion volume: 20L 6.3. 2 MS/MS operating condition See annex A. 6.3.3 LC-MS/MS determination Accord
42、ing to the estimated approximate concentration of paracetamol and saliclic acid standards in the sample solution , select the standard working solution of similar concentration to that of sample solution. The responses of paracetamol and saliclic acid standards in the sample solution should be in th
43、e linear range of the instrumental detection. The standard working solution should be injected randomlin-between the injections of the sample solution (6.2) of equal volume. Under the above instrumental condition , the retention time of paracetamol and salicylic acid standard is ca. 5. 5 and 9.0 min
44、. For the chromatogram of paracetamol and salicylic acid 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 solution can not be out of range of :t
45、O. 25min. For the same analsis 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 2 , and then the corresponding analte must be presen
46、t in the sa门lple.Table 2-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 士30:t 50 6.4 Blank te5t The operation of the blank test is the same as that described in the method of determi
47、nation, but with omiss旧nof sample addition. 7 Calculation and expression of result Calculate the residue content in the test samples according to the formula (1): x=x c x V As x m x 1 000 、，两EI/，、飞. . . . . . . . . . . . . . . . . . . . . . . 12 版权所有 禁止翻制、电子传阅、发售SN/T 1922-2007 where X-the residue co
48、ntent of analyte in the test sample , mg/kg; A-the peak area of paracetamol or salicylic acid in the test sample solution; As-the peak area of paracetamol or salicylic acid in the standard working solution; c-the concentration of paracetamol or salicylic acid in the standard working solution , ng/mL
49、; V-the final volume of sample solution , mL; m-the corresponding mass of test sample in the final sample solution , g. Note: the blank value should be subtracted from the result of calculation. 8 Li mit of determination and recovery 8. 1 Limit of determination The limits of determination of this method are: Paracetamol: 0.000 5 mg/kg; Acetylsalicylic acid (residue marker: salicylic acid): 0.
