1、中华人民共和国出入境检验检疫行业标准SN/T 1864-2007 进出口动物源食品中氯霉素残留量的检测方法液相色谱-串联质谱法Determination of chloramphenicol residue in animal-derived food for import and export-LC 2007-04-06发布2007-10-16实施中华人民共和国发布国家质量监督检验检度总局目IJ1=1 本标准的附录A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。本标准由中华人民共和国浙江出入境检验检疫局负责起草。本标准主要起草人:谢文、朱晓雨、丁慧瑛、莫君阳、黄雷芳、钱艳。本标
2、准系首次发布的出入境检验检疫行业标准。SN/T 1864-2007 1 范围进出口动物源食品中氯霉素残留量的检测方法液相色谱-串联质谱法本标准规定了动物源食品中氯霉素残留量的液相色谱串联质谱测定方法。本标准适用于虾、鱼、禽肉、肠衣和蜂蜜中氯霉素残留量的检测。2 测定方法2.1 方法提要SN/T 1864-2007 用乙酸乙醋提取样品中氯霉素，正己皖脱脂，C18固相萃取小柱净化，用液相色谱串联质谱测定和确证，同位素内标法定量。2.2 试剂和材料除另有规定外，所有试剂均为分析纯，水为二次蒸馆水。2.2.1 乙酸乙醋:高效液相色谱级。2.2.2 正己皖:高效液相色i晋级。2.2.3 甲醇:高效液相色
3、谱级。2.2.4 氧化铀。2.2.5 元水硫酸纳:6500C灼烧4h，在干燥器内冷却至室温，贮于密封瓶中备用。2.2.6 15%氧化纳水溶液:15 g氯化铀溶解于100mL水中。2.2.7 甲醇:水(2: 8，体积比)。2.2.8 氯霉素标准品(chloramphenicol，CASNO. 56-75-7 , Cll Hl2C12飞205):纯度大于等于99%。2.2.9 氯霉素d5标准品:纯度大于等于99%。2.2. 10 氯霉素标准储备溶液:称取0.0100g标准品(2.2.的，用甲醇溶解定容至100mL，溶液浓度为100g/mL 1 oC 4 oC冷藏保存。2.2.11 氯霉素而标准储备
4、溶液:称取0.0100 g标准品(2.2.9)，用甲醇溶解定容至100mL，溶液浓度为100g/mL1 oC 4 oC冷藏保存。2.2.12 标准工作曲线工作液:用甲醇:水(l: 1，体积比)稀释氯霉素标准储备溶液，5点工作曲线的浓度分别为O.05吨/mL、O.1吨/mL、O.4吨/mL、0.8ng/mL、1.2吨/mL，氯霉素d5作为同位素内标浓度为O.2吨/mLo2.3 仪器和设备2.3. 1 液相色谱串联质谱仪:配有电喷雾离子源。2.3.2 旋转蒸发器。2.3.3 粉碎机。2.3.4 均质器。2.3.5 旋涡混合器。2.3.6 固相萃取柱:C18固相萃取小柱，500mg，或相当者。甲醇和
5、水各5mL预洗。2.3.7 元水硫酸铀柱:80 mm X 40 mm (内径)筒形漏斗，底部垫5mm脱脂棉，再装50mm元水硫酸纳。SN/T 1864-2007 2.4 测定步骤2.4. 1 制样a) 虾、鱼、禽肉、肠衣:从所取全部虾、鱼或禽肉样品中取出有代表性样品约500g，用粉碎机粉碎;从所取肠衣样品中取出有代表性样品约300g，用剪刀剪碎，混合均匀，均分成两份，分别装入洁净容器作为试样，密封，并标明标记。将试样于18C冷冻保存。b) 蜂蜜:取500g代表性蜂蜜样品，未结晶的样品将其用力搅拌均匀，有结晶析出的样品可将样品瓶盖塞紧后，置于不超过60C的水浴中温热，等样品全部熔化后搅匀，迅速冷
6、却至室温。在熔化时应注意防止水分挥发。制备好的试样均分成两份，分别装入样品瓶中，密封，并标明标记。将试样室温下保存。在抽样和制样的操作过程中，应防止样品污染或发生残留物含量的变化。2.4.2 提取且)虾、鱼、禽肉:称取5g试样(精确到0.01g)置于50mL具塞离心管中，准确加入0.1mL氯霉素d5(lOng/mL)内标溶液，加25mL乙酸乙醋，在均质器中以14000 r/min均质30s，以3 000 r/min离心5mi口，将上层乙酸乙醋提取液过无水硫酸铀柱(2.3.7)，收集于浓缩瓶中，样品残渣再加入20mL乙酸乙醋，重复上述操作，合并乙酸乙醋提取溶液，在50C以下水浴减压浓缩至近干，用
7、4mL lE己皖和4mL水依次将残渣转移至10mL具塞玻璃离心管中，于旋涡混合器上以2000 r/min，混匀1mi口，以2000 r/mi口离心3mi口，弃去上层正己皖榕液，再加入4 mL正己烧，重复上述操作。b) 肠衣:称取5g试样(精确到0.01g)置于50mL具塞离心管中，准确加入0.1mL氯霉素d5(10吨/mL)内标、溶液，加25mL乙酸乙醋，于旋涡混合器上以2000 r/min，混匀1min，按上述方法操作。c) 蜂蜜:称取5g试样(精确到0.01g)置于50mL具塞离心管中，准确加入0.1mL氯霉素d5(10吨/mL)内标、溶液，加15mL 15%氧化铀水溶液(2.2.6)，加
8、25mL乙酸乙醋，于旋涡泪合器上以2000 r/min，1昆匀1mi口，以3000 r/mi口离心5min，将上层乙酸乙醋提取液过元水硫酸铀柱(2.3.7)，收集于浓缩瓶中，样品残渣再加入20mL乙酸乙醋，重复上述操作，合并乙酸乙醋提取溶液，在50C以下水浴减压浓缩至近干。用4mL水溶解残渣。2.4.3 净化将水层转移至Cl8固相萃取柱(2.3.的，再用10mL水分两次洗涤，洗涤液过Cl8固相萃取柱，弃去流出液，再用5mL甲醇水溶液(2.2. 7)洗涤，负压抽干，6mL甲醇洗脱，收集全部洗脱液，用氮气吹至约2.5mL，加水定容至5.0mL，混匀，供液相色谱串联质谱仪测定。2.5 测定2.5.1
9、 液相色谱条件a) 色谱柱:C8，5m，150mmX 4.6 mm(内径)或相当者pb) 流动相见表1; c) 流速:400L/mi川d) 进样量:20L，表1流动中目梯度洗脱时间/min水/c%)甲醇/c%)。35 65 4 75 25 5. 9 75 25 6 35 65 8 35 65 2 SN/T 1864-2007 2.5.2 质谱条件a) 离子源:电喷雾离子源;b) 扫描方式:负离子扫描;c) 检测方式:多反映监测;d) 电喷雾电压(I曰:-4 500 V; e) 雾化气压力(GSl):38 Pa; f) 气帘气压力(CUR):27 Pa; g) 辅助气流速(GS2):45Pa;
10、h) 离子源温度(TEM):525C; i) 去簇电压(DP)-65 V; j) 定性离子对、定量离子对、碰撞气能量(CE)及碰撞室出口电压(CXP)见表20表2氯霉素、氯霉素-d5定性离子对、定量离子对、碰撞气能量和碰撞室出口电压名称定性离子对定量离子对碰撞气碰撞室出口m/z m/z 能量(CE)/V电压(CXP)/V32 1. 0/256.9 16 10 32 1. 0/152. 0 25 10 氯霉素32 1. 0/256.9 32 1. 0/194.。17 10 32 1. 0/175. 8 23 10 氯霉素d5326.0/157.1 326.0/157.1 25 10 2.5.3
11、色谱测定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一起进行色谱分析。标准工作液和待测样液中氯霉素的响应值均应在仪器线性响应范围内。在上述色谱条件下氯霉素、氯霉素而的参考保留时间约为6mi口，标准溶液的选择性离子流图参见附录A中图A.102.5.4 空白试验除不加试样外，均按上述操作步骤进行。2.5.5 定性、定量测定按照液相色谱串联质谱条件测定样品和标准工作、溶液，如果检测的质量色谱峰保留时间与标准品一致，定量测定时采用同位素内标标准曲线法。定性时应当与浓度相当标准工作溶液的相对丰度一致，相对丰度允许偏差不超过表3规定的范围，则可判断样品中存在对应的被测物。表3定性确证时相对离子
12、丰度的最大允许偏差相对离子丰度允许的相对偏差2.6 结果计算和表述50% 士20%20%至50%士25%10%至20%士30%用色谱数据处理机或按式(1)计算试样中氯霉素残留含量，计算结果需扣除空白值:x _ c兰主-rn 式中:X 试样中氯霉素的残留量，单位为微克每千克(g/kg);c 从标准曲线上得到的氯霉素溶液浓度，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);m 最终样液代表的试样质量，单位为克(g)o三二10%士50%. ( 1 ) 3 SN/T 1864-2007 3 测定低限(LOQ)和回收率3.1 测定低限(LOQ)测定低限为o.1g/kg(LOQ
13、)。3.2 回收率回收率的实验数据(在不同添加浓度范围内)见表40表4回收率回收率/C%) 添加浓度/C!-g/kg)虾鱼肠衣0.1 70. 089.。89. O 108.。82. O 106.。0.4 80. 0110. 0 72. 5105. 0 75. 0110. 0 O. 8 72. 5105. 0 81.2107.5 85. 0110. 0 4 禽肉蜂蜜81. O 110.。93. O 110.。75. 0110. 0 72. 5110. 0 77. 5108. 7 72. 5103. 7 SN/T 1864-2007 附录(资料性附录)氯毒素标准品选择性离子流固A 3068J 32
14、 1. 0/152.0 00 80 00 22 二三【552一7.0 7.5 6.01 3. 05凹:1.:18:1.68 4.:12、4.471.672田5.12).2巧，4._ 7巧气17,6.46,6.68 7.25 7.60 J76 J一户_._.3.0 3.5 1.0 1.5 5.0 5.5 6.0 6.5 7.0 7.5 l/lllll1 321. Oj256. 9 一二-_2.0 2.5 3.0 3.5 1.0 l/lllll1 1. 5 1. 0 0.5 (三【5526.01 12 1. 0/194.0 3.193.15，3.513.9巴。但11.311.64.705:055.
15、31.5.52 5.HO) .6.276.飞6.547.46、7.61一, 一一一一一3.0 3.5 1.0 1.5 5.0 5.5 6.0 6.5 7.0 7.5 l/lllll1 2.5 14HO 1000 -1 o-_-一、-_一-丁一一-一一-0.5 1. 0 1. 5 2.0 2.5 (三【552ULJJPEOUq1UTV 12 1. 0/175.8 3.07,3.13 3.38 . .:1 .47 1.10 4.251.17 ).60 5O 一_-一牛斗-3.0 3.5 1.0 1.5 5.0 l/lllll1 9的0500 (三【552业LJL巴126.0/157.1 2.5 2
16、.0 1. 5 1. 0 0.5 才5000斗 ，元巳、，-司一气-叮-一0.5 3.D)、3.18).31,. 3.52 3.91 1.39、4.524.751.953.0 3.5 1.0 1.5 5.0 l/lllll1 2.5 2.0 1. 5 1. 0 5 氯霉素(0.05ng/mL)和氯霉素-d5(0.2ng/mL)标准品的选择性离子流图图A.1SN/T 1864-2007 Foreword Annex A of this standard is an informative annex. This standard was proposed byand is under the c
17、harged of certification and accreditation administa tion of the People s Republic of China. This standard was drafted by Zhejiang Entry-Exit Inspection and Ouarantine Bureau of the People s Republic of China. The standard was mainly drafted by Wen Xie ,Xiao-yu Zhu , Hui-yin Ding ,Jun-yang Xi , Lei-f
18、ang Huang , Yan Oian. This standard is a professional 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. 6 SN/T 1864-2007 Determination of chloramphenicol residue in animal-deriv
19、ed food for import and export LC-MSjMS method 1 Scope The standard specifies the methods of determination bLC-MS/MS of chloramphenicol in animal-de rived food. This standard is applicable to the determination of chloramphenicol in shrimp, fish , poultry meat , cas ing and honesamples. 2 Method of de
20、termination 2. 1 Principle Chloramphenicol is extracted from the sample bethI acetate. It was defatted with n-hexane and cleaned up with C1日column. Finallit is determined and confirmed bLC-MS/MS. Internal standard method is used. 2. 2 Reagents and materials Unless otherwise specified , all reagents
21、used should be analytical grade, water is double distilled water. 2.2. 1 Ethyl acetate: HPLC grade. 2.2.2 n-Hexane: HPLC grade. 2.2.3 Methanol: HPLC grade. 2. 2. 4 Sodium chloride. 2.2.5 Anhydrous sodium sulphate: Ignite for 4 h at 650C , cool to room temperature in desiccator and keep in a tightly
22、closed container. 2.2.6 15% sodium chloride solution: Dissolve 15 9 sodium chloride in 100 mL water. 7 SN/T 1864-2007 2.2.7 Methanol: water 2 : 8( V / V) 2.2.8 Chloramphenicol (CAS NO. 56-75-7 , C H2CI2N205): Purity关99%.2.2.9 Chloramphenicol-d5: Purit;?:99%. 2.2.10 Stock standard solution of chloram
23、phenicol:Accurately weigh 0.0100 9 standard (2.2. 7), dissolve with methanol and quantitatively on 100 mL volumetric flask , the concentration of solution is 100g/mL，should be stored at 10C .40C in refrigerator. 2.2. 11 Stock standard solution of chloramphenicol-d5: Accurately weigh O. 010 0 9 stand
24、ard (2.2.酌，dissolvewith methanol and quantitatively on 100 mL volumetric flask , the concentration of solution is 100g/mL. should be stored at 10C .40C in refrigerator. 2.2.12 Calibration curve standard working solutions: Working solutions of CAP were prepared in methanol:water 1 1 (V/V) at five con
25、centration levels, O. 05 ng/mL, O. 1 ng/mL, O. 4 ng/mL, 0.8 ng/mL, 1.2 ng/mL,with CAP-d5 as internal standard at a concentration 0.2 ng/mL. 2.3 Apparatus and equipment 2.3. 1 Li quid chromatography combined with electrospraionization mass spectrometr. 2.3. 2 Rotary vacuum evaporator. 2.3.3 Blender.
26、2.3.4 Homogenizer. 2.3. 5 Vortex mixer. 2.3.6 C8 column: 500 mg , or equivalent. It was conditioned with 5 mL methanol followed by 5 mL water. 2.3. 7 Column of anhydrous sodium sulfate: 80 mm x 40 mm (i. d) cylinder funnel , pack with ca 5 mm absorbent cotton at the bottom of the column and fill in
27、50 mm anhdrous sodium sulfate. 2.4 Procedure 2.4. 1 Preparation of test sample a) Shrimp, fish , poultry meat and casing: Take the representative portions from the whole primary shrimp or fish or poultry meat sample. It is about 500 9 and ground in a blender. Casing sample is about 300 g , a small c
28、ut made with scissors. Keep the prepared sample into two sample bottles , 8 SN/T 1864-2007 seal and labe l. The test sample is stored in - 1SoC refrigerator. b) Hone: Honey sample is about 500 g. The sample that is not crystallized shall be stirred well to make homogeneous. If the sample is crystall
29、ized , it must be warmed in a water-bath below 600C with the sample bottle covered tightl，mix thoroughly when all sample has melted, then cool im mediately to room temperature. In the course of melting the sample , precautions must be taken to avoid evaporation of water from the sample. Keep the pre
30、pared sample into two sample bot tles , seal and label. The honey test sample is stored at room temperature. In the course of sampling and sample preparation , precautions must be taken to avoid contamina tion or anfactors that macause the change of residue content. 2.4. 2 Extraction a) Shrimp, fish
31、 and poultrmeat: Weigh ca 5 9 of the test sample (accurate to O. 01 g) into a 50 mL centrifuge tube,add O. 1 mL CAP-d5(10 ng/mL) and 25 mL ethyl acetate. Homogenize for 30 s at 14000 r/min. Centrifuge for 5 min at 3 000 r/min. The supernatant layer was passed , through an hydrous sodium sulfate colu
32、mn into flask. Repeat the extraction of the residues in the same way with 20 mL ethyl acetate and combined the solution. The solution evaporate to nearly dryness in a water bath below 50oC. Add 4 mL n-hexane and 4 mL water to dissolve residues , transfer the solution into 10 mL graduated glass tube.
33、 81end for 1 min at 2 000 r/min , centrifuge for 3 min at 2000 r/min ,discard supernatant layer. Add 4 mL n-hexane and repeat the procedure. b) Casing: Weigh ca 5 9 of the test sample (accurate to 0.01 g) into a 50 mL centrifuge tube, add 0.1 mL CAP-d5(10 ng/mL) and 25 mL eth1 acetate. 81end for 1 m
34、in at 2 000 r/min ,operate the above procedure. c) Honey:Weigh ca 5 9 of the test sample (accurate to 0.01 g) into a 50 mL centrifuge tube ,add 0.1 mL CAP-d5(10 ng/mL) , 15% sodium chloride solution and 25 mL eth1 acetate. 81end for 1 min at 2 000 r/min. Centrifuge for 5 min at 3 000 r/min. The supe
35、rnatant layer was passed through anhdrous sodium sulfate column into flask. Repeat the extraction of the residues in the same way with 20 mL ethyl acetate and combined the solution. The solution evaporate to nearly drness in a water bath below 50oC. Add 4 mL water to dissolve residues. 2.4.3 Clean u
36、p Transfer the above water solution into the C18 column. Wash the graduated glass tube two times with 5 mL water,one time with 5 mL methanol solution (2.2.7) ,pass washes through C1B column ,and dis card the eluted solution. The cartridge is evacuated continuously to dryness under vacuum. Elute the
37、column with 6 mL methanol and collect eluted solution. The above solution is under a gentle stream of nitrogen gas to 2. 5 mL. Adjust volume of eluate to 5.0 mL with water. The solutions used for LC-MS/MS determination. 9 SN/T 1864-2007 2. 5 Determination 2. 5. 1 HPLC operating conditions a) Column:
38、 ZORBAX Eclipse XDB-Cs ，5m， 150 mm x 4.6 mm(i. d) ,or the equivalent; b) Mobile phase: table 1 ; c) Flow rate: 400L/min; d) Injection volume:20L Table 1-Gradient of mobile phase Time/min Water/ (%) Methanol/ ( % ) 。35 65 4 75 25 5.9 75 25 6 35 65 8 35 65 2. 5. 2 Mass spectral acquisition a) Source:E
39、SI; b) Polarity: Negative; c) Mode:Multiple reaction monitoring; d) IS:一4500V; e) GSI:38 Pa; f) CUR:27 Pa; g) GS2 : 45 Pa; h) TEM:5250C; i) DP:一65V; j) Transitions for confirmation and quantification , CE , CXP sees table 2. 10 SN/T 1864-2007 Table 2-Transitions for confirmation and quantification,C
40、E ,CXP Transitions for Transitions for Compound confirmation quantification CE/V CXP/V m/z m/z 321.0/256.9 16 10 321.0/152.。25 10 CAP 321.0/256.9 321.0/194.。一1710 321.0/175.8 23 10 CAP-d5 326.0/157.1 326.0/157.1 25 10 2. 5. 3 LC-MS/MS determination According to the approximate concentration of analt
41、e in sample solution , select the standard work ing solution with similar responses to that of sample solution. The responses of the analte in the standard working solution and the sample solution should be within the linear range of the instrument detection. Under the above LC-MS/MS operating condi
42、tion, the retention time of CAP is about 6 min , selected ion chromatograms of the standards see Figure A. 1 in annex A. 2. 5. 4 81ank test The operation of the blank test is the same as the described in the method of determination, but with the omission of sample addition. 2.5.5 Confirmation of LC-
43、MS/MS Under LC-MS/MS conditions, the working solution and sample solution is injected. If the retention times of sample chromatogram peaks are consistent with that of standard solution , calibration curve method with isotope internal standard is used for quantitative measurement. The relative intens
44、ities of sample transitions shall correspond to those of standard solution transitions for confirmation. The concentration of standard solution should be same with those of sample solution. The permitted tol erances listed in table 3 , then the corresponding analte must be present in sample. Table 3
45、-Maximum permitted tolerances for relative ion intensities while confirmation Relative intensity 50% Permitted tolerances 士20%2. 6 Calculation and expression of result 20% to 50% 士25%10% to 20% 士30%10% :t 50% Calculation the content of CAP residue in the test sample bLC-MS/MS data processor or accor
46、ding to the formula (1) The blank value should be subtracted from the above result of calculation. 11 SN/T 1864-2007 X二些兰厅7、，两EI/，、飞. . . . . . . . . . . . . . . . . . . . . . . . . . . . . Where , X-the residue content of CAP in the test sample，g/kg; c-the concentration of CAP is from calibration c
47、urve, ng/mL; V-the final volume of the sample solution , mL; m-mass of test sample of final sample solution , g. 3 Li mit of quantification CLOQ) and recover 3. 1 Li mit of quantification The limit of quantification for CAP is O. 1g/kg. 3. 2 Recovery According to the experimental data , the correspo
48、nding recoveries of fortifying concentrations see table 4. Table 4-Recovery Recovery / (%) Spike/ (g/kg) Shrimp Fish Casing Poultry meat Honey 0.1 70.0-89.。89.0-108.。82.0-106.。81.0-110.。93.0-110.。0.4 80.0-110.。72.5-105.。75.0-110.。75.0-110.。72.5-110.。0.8 72.5-105.。81.2-107.5 85.0-110.。77.5-108.7 72.5
49、-103.7 12 SN/T 1864-2007 Annex A (informative annex) Selected ion chromatograms of CAP and CAP-d5 standards 3068J 32 1. 0/152.0 00 80 00 22 二三【552一7.0 7.5 6.01 3. 05凹:1.:18:1.68 4.:12、4.471.672田5.12).2巧，4._ 7巧气17,6.46,6.68 7.25 7.60 J76 J一户_._.3.0 3.5 1.0 1.5 5.0 5.5 6.0 6.5 7.0 7.5 l/lllll1 321. Oj256. 9 一二-_2.0 2.5 3.0 3.5 1.0 l/lllll1 1. 5 1. 0 0.5 (三【
