1、中华人民共和国出入境检验检疫行业标准SN/T 1627-2005 进出口动物源食品中硝基映喃类代谢物残留量测定方法高效液相色谱串联质谱法Determination of metabolites of nitrofurans residues in animal origin food for import and export-HPLC-MS/MS 2005心8-18发布. ZE-EE E- . , 削肌EEEE-E咛iEEE啤啤nuul-EE- i: MHHHUm -呻呻呻叫MMMMHnumwmm nhu -呻川川Hot-nhU HHHHHHHnu - i . i EE- 2006-02-0
2、1实施中华人民共和国发布国家质量监督检验检疫总局前言本标准附录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。本标准起草单位:中华人民共和国河北出入境检验检疫局。本标准主要起草人:王凤池、马振栋、郭春梅、陈瑞春、窦彩云。本标准系首次发布的出入境检验检疫行业标准。SN/T 1627-2005 I SN/T 1627一20051 范围进出口动物源食晶中硝基映喃类代谢物残留量测定方法高效液相色谱串联质谱法本标准规定了动物源食品中硝基映喃类代谢物残留量液相色谱质谱联用测定方法。本标准适用于虾、鸡肉、蜂蜜、肠衣中硝基映喃类代谢物残留量的测定。2 测定方法2. 1 方法提要样品中与蛋
3、白质结合的硝基映喃类代谢物(参见附录A)在酸性环境下水解游离出来，用邻硝基苯甲醒衍生后，以乙酸乙醋提取。经过净化浓缩，用液相色谱质谱联用仪测定。外标法定量。2.2 试剂和材料除另有规定外，所用试剂均为分析纯，水为超纯水。2.2. 1 乙睛:HPLC级。2.2.2 甲醇:HPLC级。2.2.3 乙酸乙醋:HPLC级。2.2.4 邻硝基苯甲醒。2.2.5 二甲亚矶。2.2.6 元水硫酸铀:650C灼烧4h，冷却后贮于密闭容器中备用。2.2.7 盐酸溶液:0.125mol/L。2.2.8 氢氧化锅溶液:1 mol/L。2.2.9 氢氧化铀溶液:0.1mol/L。2.2.10 氯化纳溶液:300g/L
4、。2.2. 11 盐酸溶液:1 mol/L。2.2. 12 衍生化试剂z称取100mg邻硝基苯甲醒溶于12.5mL二甲亚枫中，现用现配。2.2. 13 定容液:0.1%乙酸水溶液z乙睛z甲醇3: 3 : 2(体积分数)。2.2. 14 硝基映喃类代谢物标准品:AOZ、AMOZ、AHD.HCLSEM HCl，纯度注99.0%。2.2.15 标准储备液z分别称取硝基峡喃类代谢物标准品AOZ、AMOZ10. 0 mg , AHD HCl 13.2 mg ,SEM HCl 14.9 mg(准确至0.1mg).分别用甲醇榕解并转移至20mL容量瓶中，用甲醇定容至刻度，该溶液浓度为0.5mg/mL.40C
5、以下保存。2.2.16 标准中间液:分别吸取四种硝基映喃类代谢物的标准储备液各l.0 mL，用甲醇定容至50mL , 浓度为10g/mL。2.2.17 标准工作液:取标准中间液l.0 mL.甲醇稀释定容至100mL，浓度为0.1g/mL。2.2. 18 校正曲线标准工作液:将标准工作液稀释至适宜浓度制作校正曲线，临用时配制。2.3 仪器和设备2.3.1 液相色谱质谱联用仪，可进行二级质谱分析。2.3.2 电子天平。2.3.3 超声波清洗器。1 SN/T 1627-2005 2.3.4 恒温水浴振荡器。2.3.5 pH t十。2.3.6 自动平衡离心机(最大转速5000 r/min)。2.3.7
6、 旋转蒸发器。2.3.8 氮吹仪。2.3.9 均质器。2.3. 10 游涡混合器。2.4 测定步骤2.4.1 样晶处理蜂蜜样品混匀后可直接称取。肉类和虾类先用绞肉机绞碎，混匀。盐横肠衣样品先用剪刀剪成小于5mm宽的段，用清水洗去盐分，再用蒸锢水冲洗，在丝网器皿上摊开沥去水分5mino 2.4.2 水解、衍生和提取称取1g(精确至0.01g)混匀后的样品于50mL磨口具塞离心管中，加入30mL O. 125 mol/L盐酸溶液(2.2.7)和1.0 mL衍生试剂(2.2.12) ，肉类和虾样品均质1min，蜂蜜和肠衣样品盖好塞子用力摇匀，松开塞子放气。盖好塞子后置于恒温水域振荡器上370C振荡水
7、解16小时(过夜)。2.4.3 净化向水解榕液中加入约4.0 mL 1 mol/L氢氧化铀榕液(2.2.的，用1mol/L盐酸溶液(2.2.11)和0.1 mol/L氢氧化铀溶液(2.2.9)，调节pH值至7.0 7. 50 3 000 r / min离心10min，上清液倾入250 mL分液漏斗中。残渣用5mL水洗涤，用玻璃棒搅匀，3000r/min离心10min，上清液并入分液漏斗中。向分液漏斗中加入40mL乙酸乙醋，振摇1min，静置3min以上。分层后将下层水相转移至另一分液漏斗中，用40mL乙酸乙醋再提取一次，弃去水相。用10mL 300 g/L氯化纳榕液(2.2.10)依次洗涤两分
8、液漏斗中的乙酸乙醋洛液，弃去水相。将乙酸乙醋层，通过装有2g无水硫酸铀的漏斗过滤至鸡心瓶中。500C旋转蒸发至3mL5 mL，转移至12mL试管中，用5mL乙酸乙醋分3次洗涤鸡心瓶，合并洗涤液于试管中，然后在500C氮气流下吹干。用定容被(2.2.13)定容至1.0 mL，通过0.45m滤膜过滤至样品瓶中，供液相色谱质谱测定。2.4.4 测定2.4.4. 1 液相色谱条件a) 色谱柱:Cl8 , 150 mmX2.1 mm(内径)，填料颗粒直径5m;b) 流动相:A为0.1%乙酸水溶液，B为乙腊梯度洗脱程序见表1。表1梯度洗脱程序时间/minA/C%) B/C%) 。75 25 10 30 7
9、0 13 10 90 平衡时间为6min。c) 流速:0.3mL/min; d) 色谱柱温度:250C;e) 进样量:15L。2 SN/T 1627-2005 2.4.4.2 质谱条件a) 离子源ESI，正模式;b) 雾化喷嘴压力:35.0psi; c) 干燥气流量:9.0L/min; d) 干燥气温度:350.C ; e) 毛细管电压:AMOZ，SEM，AHD，AOZ分别为3861V , 3713 V , 4500 V.3516 V; f) 撇取器电压:AMOZ，SEM，AHD，AOZ分别为15.0V , 15.0 V ,15.0 V , 19.2 V; g) 碰撞电压:1.0 V。2.4.
10、4.3 色谱测定根据试样中被测物的含量情况，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硝基映喃类代谢物的响应值均应在仪器线性响应范围内。对标准工作液和样液等体积参插进样测定。上述色谱条件下，四种硝基映喃类代谢物的保留时间，母离子和子离子见表2。标准品总离子流图参见附录B。表2四种被测物的保留时间、母离子和子离子被测物AMOZ SEM AHD AOZ 保留时间/min1. 72 5.62 6.22 7.55 母离子(m/z)335 209 249 236 子离子Cm/z)291、262166、192134、178134、1042.4.5 空白实验除不称取样品外，均按上述测
11、定条件和步骤进行。2.5 结果计算和表述2.5.1 定性测定进行样品测定时，如果检出的质量色谱峰保留时间与标准样品一致，并且在扣除背景后的样品谱图中，各定性离子的相对丰度与浓度接近的同样条件下得到的标准溶液谱图相比，误差不超过表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丰度的最大允许误差相对离子丰度50% 20%至50%10%至20%:(10% 允许的相对误差土20%土25%土30%土502.5.2 定量测定用数据处理软件中的外标法，或按式(1)计算试样中硝基映喃类代谢物的残留量zV . P -m 飞/-。A-s 二AA-J，、-X . C 1 ) 式中:X一一试样中
12、硝基吱喃类代谢物的残留量，单位为微克每千克(g/kg); V一一样液最终定容体积，单位为毫升CmL);A一一样液硝基峡喃类代谢物的色谱峰面积，单位为平方毫米Cmm2); Ao -空白实验硝基映喃类代谢物的色谱峰面积，单位为平方毫米Cmm2); As一一标准工作液硝基峡喃类代谢物的色谱峰面积，单位为平方毫米Cmm2); c 标准工作液中硝基映喃类代谢物的浓度，单位为纳克每毫升Cng/mL); 3 SNjT 1627-2005 m-一最终样液代表的试样量，单位为克(g)。3 测定低限、回收率3. 1 测定低限本方法的测定低限为:1g/峙。3.2 回收率3.2. 1 虾中回收率的实验数据添加浓度在1
13、g/kg、2g/kg、5g/同时，AMOZ回收率在76.5% 79.3%; SEM回收率在83. 2%107. 9% ;AHD回收率在80.6%95. 1% ，AOZ回收率在73.2%96. 4%。3.2.2 鸡肉中回收率的实验数据添加浓度在1g/kg、2g/kg、5g/kg时，AMOZ回收率在66.5% 71. 7%; SEM回收率在86. 3%93. 3% ;AHD回收率在72.2%79. 7% ，AOZ回收率在73.3%91. 4%。3.2.3 蜂蜜中回收率的实验鼓据添加浓度在1g/kg、2g/kg、5g/kg时，AMOZ回收率在78.3% 82. 8%; SEM回收率在80. 5%98
14、. 9% ;AHD回收率在66.6%89. 3% ，AOZ回收率在78.6%99. 5%。3.2.4 肠衣中回收率的实验敢据添加浓度在1g/kg、2g/kg、5g/同时，AMOZ回收率在63.6% 84. 5%; SEM回收率在81. 0%90. 4% ;AHD回收率在70.2%77. 9% ，AOZ回收率在66.0%85. 0%。4 SN/T 1627-2005 附录A(资料性附录)硝基映喃类代谢物中英文名称和缩写对照表表A.1原药代谢物中文名称代谢物英文名称代谢物名称缩写CAS编号映喃瞠酣3氨基-2-p恶略炕酣3-Amino-2-oxazolidinone AOZ 80-65-9J 峡喃它
15、酣5-吗琳基甲基-3氨基5-morpholino- methyl-3-amino AMOZ 43056-63-9 J 2-略略炕酣-2-oxazolidinone 峡喃西林氨基腺Semicarbazid SEM 563-41-7J 峡喃妥因1氨基海特因1-aminohydantoin AHD 2827-56-7J 5 SN/T 1627-2005 附录B(资料性附录)硝基睐喃类代谢物标准晶衍生物总离子流固3 tMU4 huSA hx 2 。2 4 6 8 10 Time/min 注:图中1，2，3，4各峰分别为AMOZ，SEM、AHD、AOZ的衍生物。固B.1 硝基映喃类代谢物衍生物的TIC固
16、6 SN/T 1627-2005 Foreword Annex A and annex B of this standard is an informative annex. This standard was proposed by and is under the charge of National Regulatory Commission for Certi fication and Accreditation. This standard was drafted by Hebei Entry-Exit Inspection and Quarantine Bureau of the
17、Peoples Re public of China. The main drafters of this standard are Wang fengchi , Ma zhendong , Guo chunhai , Chen ruichun and Dou caiyun. This standard is a Entry-Exit Inspection and Quarantine professional standard published for the first time. 7 SN/T 1627-2005 Determination of the metabolites of
18、nitrofurans residues in animal origin food for import and export-HPLC-MS/MS 1 Scope This standard specifies the method of determination of the metabolites of nitrofurans residues in ani mal origin food by HPLC-MS/MS. This standard is applicable to the detemination of the metaboltes of ntrofurans res
19、idues in shrimp, chicken , honey, salted casing. 2 Method of determination 2. 1 Abstract of this method Metabolites of nitrofurans(see annex A) in sample are hydrolised with O. 125 mol!L HCI and deriva tised with 2-nitrobenzaldehde. The solution are extracted with Ethyl acetate. Finally the ready so
20、lu tion are determined by LC-MS/MS, using external standard method. 2. 2 Reagents and mater ials AII reagents used are A. R. and water is deionized water unless specfcally noted. 2.2. 1 Acetonitrile: HPLC grade. 2. 2. 2 Methanol: HPLC grade. 2. 2. 3 Ethyl acetate: HPLC grade. 2.2.4 2-Nitrobenzaldehy
21、de. 2. 2. 5 Dimethyl sulfoxide. 2.2.6 Anhydrous sodium sulfate: Ignite at 650 c for 4 h,and keep in a tightly closed container af ter cool. 2.2.7 Hydrochloric acid: 0.125 mol!L. 2. 2. 8 Sodium hdroxide: 1 mol/L. 8 SN/T 1627-2005 2.2.9 Sodium hydroxide: 0.1 mol/L. 2.2.10 Sodium chloride: 300 g/L. 2.2
22、.11 Hydrochloric acid: 1 mol/L. 2.2.12 Derivatization reagent: weigh 100 mg 2-nitrobenzaldehyde dissolved with 12.5 mL dimethyl sulfoxide, fresh prepared dail. 2.2.13 Dissolve solution:O. 1 % acetic acid : acetonitrile : methanol3 : 3 : 2(v/v). 2. 2. 14 Standard of metabolites of nitrofuran: AOZ、AMO
23、Z、AHD HCI、SEM.HCI、purity99. 0%. 2.2.15 Stock standard solution: accurately weigh AOZ、AMOZ10.0 mg , AHD HCI 13. 2 mg、SEM HCI 14.9 mg standard(2. 2. 14) (accurate to O. 1 mg) ,dissolve with methanol and dilute to the scale in a 20 mL volumetric flask individuall. The concentration of solution is O. 5
24、mg/mL, should be stored at 1 t -4 oC in refrigerator. 2.2.16 Mixed Standard middle solution: seperately suck 1.00 mL stock standard solution (2.2.15) into a 50 mL volumetric flask,dilut with methenol to scale and mix well. The concentration of solu tion is 10.0g/mL. 2.2.17 Standard working solution:
25、 suck 1.00 mL middle standard solution (2.2. 16) into a 100 mL volumetric flask, dilut with methenol to the scale and mix well. The concentration of solution is 0.1g/mL. 2.2.18 Calibration curve standard working solution : according to the concentration required , dilut standard working solution (2.
26、2. 17)with mobile phase. 2.3 Apparatus and equipment 2.3.1 High performance liquid chromatography tandem mass spectrograph. 2. 3. 2 Electronic balance. 2.3.3 Ultrasonic cleanser. 2. 3. 4 Shakeing waterbath . 2.3.5 pH meter. 9 SN/T 1627-2005 2.3.6 Centrifuge: the max speed 5 000 r/min . 2. 3. 7 Rotar
27、y vacuum evaparator. 2.3.8 Nitrogen evaparator. 2.3.9 Homogenizer. 2.3. 10 Vortex minishaker. 2. 4 Procedure 2. 4. 1 Preparation of test sample Honesamples can be weighed directly after mixed. Meat and shrimp samples should be smashed by meat chopper. Salted casing samples should be scissored to pie
28、ces less than 5 mm, then washed with water and fi nally droped for 5 min. 2. 4. 2 Extraction Weigh臼1.0 9 of test sample (accurate to O. 01 g) into a 50 mL centrifuge tube , add 30 mL O. 125 mol/L HCI (2.2.7) and 1. 0 mL derivatised reagent (2.2. 12) , meat and shrimp homogenized 1 min , honey and sa
29、lted casing strongly shaked after cap , degas. Shake for 16 hrs(over night) at 37 t in a shaking waterbath. 2. 4. 3 Clean-up Add 4.0 mL sodium hdroxide to the sample solution , adjust pH to 7.0-7.5 with 1 mol/L hydro chloric acid ( 2.2. 11) and O. 1 mol/L sodium hydroxide (2.2.9). Centrifuge for 10
30、min at 3000 r/min . Transfer the solution into a 250 mL separator funnel ,wash the centrifuge tube with 5 mL water, mixed round with galss stick, centrifuge at 3 000 r/min for 10 min,combine the solution to the separetor funnel. Add 40 mL ethyl acetate to the separator funnel ,shake 1 min, let stand
31、 for separating more than 3 min , tansfer the water phase into another separator funnel , add another 40 mL ethI acetate and extraced once more. Wash the organic layer addtionally with 10 mL sodium chloride (2.2. 10). Transfer the ethyl acetate layer in a 250 mL Erlenmeyer flask by passing through a
32、 funnel which is filled with ca. 2g anhdrous sodium sulfate (2. 2. 6). Evoparate the ethyl acetate with Rotovap at ca. 50 t until3 mL-5 mL remained. Tansfer the remains into a 12 mL glass tube . Wash the flask 3 times with 5 mL ethyl acetate , blow to dryness at 50 t under light nitrogen. The residu
33、e is dis solved with 1.0 mL dissolve solution(2. 2.13) in ultrasonic bath and mix well by vortex minishaker, filtered through 0.45m membrane and ready for LC-MS/MS determination. 10 SN/T 1627-2005 2. 4. 4 Determination 2.4.4.1 HPLC operating condition a) Column: C，日150mm x 2.1 mm(i. d.) ，5m particle
34、 size; b) Mobile phase: A: 0.1% acetic acid ,B: acetontrile, The gradient program is showed in table 1. Table 1 gradient program Time/min A/(%) B/C%) 。75 25 10 30 70 13 10 90 balance time 6 min. c) Flow rate: 0.3 mL/min; d) Column temperature: 25 c ; e) Injection volume: 15L 2.4.4.2 MSnditions: a) l
35、on Source Type: ESI , positive mode; b) Nebulizer: 35.0 psi; c) Dry Gas: 9.0 L/min; d) Dry Temp: 350 oC; e) Capillary: for AMOZ , SEM , AHD , AOZ is 3861 V,3713 V,4500 V,3516 V respectively; f) Skim: for AMOZ , SEM , AHD , AOZ is 15.0 V. 15.0 V, 15.0 V, 19.2 V respectively; g) Fragmentation Ampl: 1.
36、0 V. 2. 4. 4. 3 LC-MS/MS determination According to the approximate concentration of analyte 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 wi
37、thin the linear range of the instrument detection. The mixed standard working solution and the sample solution should be injected with e qual volume alternatively. Under the above operating condition, the retention time, parent ions and daughter ions seeing the table 2, For the chromatogram of the s
38、tandard ,see annex B. Table 2 The retention time ,parent ions and daughter ions of analyte Analyte AMOZ SEM AHD AOZ Retention/min 1.72 5.62 6.22 7.55 Parent ions 335 209 249 236 Daughter ions 291、262166、192134、178134、10411 SN/T 1627-2005 2. 4. 5 Blank test The operation of the blank test is the same
39、 as that described in the method of determination , but with the omission of sample addition. 2.5 Calculation and expression of result 2.5. 1 Confirmation If the retention times of sample chromatogram peaks are consistent with the standards , and after background compensation , the relative ion inte
40、nsity shall correspond to those of the calibration standard , at comparable concentrations , measured under the same conditions, within the tolerances listed in table 3, then the corresponding analte must be present in the sample. Table 3 Maximum permitted tolerances for relative ion intensities whi
41、le confirmation Relative intensity 50% 20% to 50% 10% to 20% :0( 10% permitted tolerances 士20%土25%土30%士502. 5. 2 Determination of quantitative Calculate the content of analte in test sample bdata processor or according to formula (1). (A - Ao) C V x= ( 1 ) As. m where: X一一-theresidue of the analyte
42、in test sample，g/kg; V-the final volume of the sample solution ,mL; A一一-thepeak area of analte of the sample solution , mm2; Ao一一thepeak area of blank test , mm飞As-the peak area of analyte of the standard solution , mm勺C一一-theconcentration of nitrofuans in the standard solution，g/mL; m-the correspon
43、ding sample weight of the final solution , g. 3 Limit of determination and recovery 3. 1 Limit of determination The limit of determination of this method is 1g/kg. 3. 2 Recovery 3. 2. 1 Recoverof fortifying in shrimp sample The recovery range of AMOZ is 76. 5% -79.3%; SEM is 83. 2% -107. 9% ; AHD is
44、 80. 6% -95. 1 % , AOZ is 73. 2% -96.4% , when fortified at the concentration of 1g/kg， 2g/kg， 5g/kg. 12 SN/T 1627-2005 3. 2. 2 Recovery of fortifing in chichen sample The recovery range of AMOZ is 66. 5% -71. 7%; SEM is 86.3% -93.3%; AHD is 72. 2% -79. 7% , AOZ is 73.3% -91. 4% , when fortified at
45、the concentration of 1g/阔，2g/kg， 5g/kg. 3. 2. 3 Recovery of fortifing in honey sample The recovery range of AMOZ is 78. 3% -82.8%; SEM is 80. 5% -98.9%; AHD is 66.6% -89.3% , AOZ is 78. 6% -99.5% , when fortified at the concentration of 1g/kg， 2g/kg， 5g/kg. 3. 2. 4 Recoverof fortifying in salted臼sin
46、gsample The recovery range of AMOZ is 63. 6%-84. 5%; SEM is 81. 0% -90. 4%; AHD is 70. 2%-77.9% , AOZ is 66.0 % -85.0 %, when fortified at the concentration of 1g/kg， 2g/kg， 5g/kg. 13 SN/T 1627-2005 Parent drug Furazolidone Furaltadone Nitrofurazone Nitrofurantion 14 Annex A (informative annex) Nitr
47、ofuran drugs and its corresponding metabolites Table A.1 Metabolite Ab. 3-Amino-2-oxazolidinone AOZ 5-morpholino-methyl-3-amino AMOZ -2-oxazolidinone Semicarbazid SEM 1-aminohydantoin AHD CAS.NO. 80-65-9J 43056-63-9J 563-41-7J 2827-56-7J SN/T 1627-2005 Annex B (informative annex) Total ion chromatog
48、ram of nitrofurans metabolite derivative standards lMu4 huA hx 4 3 2 3 。2 4 6 8 10 Time/min Where: The peak No. 1.2.3.4. is corresponding to the derivative of AMOZ.SEM.AHD.AOZ respectively Fig. B. 1 Total ion chromatogram of nitrofurans metabolite derivative standards BON-hN户同军中华人民共和国出入境检验检疫行业标准进出口动物源食晶中硝基眼睛类代谢物残留量测定方法高效液相色谱串联质谱法SN/T 1627-2005 * 中国标准出版社出版发行北京复兴门外三里河北街16号邮政编码:100045网址电话:6852394668517548 中国标准出版社秦皇岛印刷厂印刷* 印张1.25 字数23千字2005年11月第一次印刷开本880X 1230 1/16 2005年11月第一版* 定价12.00元如有印装差错由本社发行中心调换版权专有侵权必究举报电话:(010)685335
