1、中华人民共和国出入境检验检疫行业标准SN/T 1626-2005 进出口肉及肉制品中甲硝略、替硝睡、奥硝略、罗硝瞠、二甲硝咪略、塞克硝瞠残留量测定方法高效液相色谱法Determination of metronidazole, tinidazole , ornidazole , ronidazole, dimetridazole and secnidazole residues in meat and meat products for import and export-HPLC 2005-08-18发布060608000108 2006-02-01实施中华人民共和国发布国家质量监督检验检瘦
2、总局前言本标准的附录A为资料性附录。本标准由国家主人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、郭春海、吕红英、艾连峰。本标准系首次发布的出入境检验检疫行业标准。SN/T 1626-2005 I 1 范围进出口肉及肉制品中甲硝睡、替硝瞠、奥硝睡、罗硝瞠、二甲硝咪瞠、塞克硝瞠残留量测定方法高效液相色谱法SN/T 1626-2005 本标准规定了肉及肉制品中罗硝唾、二甲硝咪哇及其共同的代谢物、替硝略、奥硝略、塞克硝瞠、甲硝瞠7种硝基咪瞠残留量的高效液相色谱测定方法。本标准适用于鸡肉、猪肉中罗硝瞠、二甲硝咪瞠及其共同的代谢物、
3、替硝略、奥硝略、塞克硝瞠、甲硝哇残留量的测定。2 样晶处理将实验室样品用绞肉机全部绞碎。样品量太大时从每块样品上取代表性的部分，然后全部绞碎，混合均匀后，分为两份，一份进行测定，另一份装人密闭容器内，-180C冷冻避光保存。3 测定方法3. 1 方法提要试样中的硝基咪瞠残留用乙酸乙醋提取，通过SCX柱净化后，用配有紫外检测器的高效液相色谱仪测定，外标法定量。3.2 试剂和材料除另有规定外，所用试剂均为HPLC级，水为高纯水。3.2. 1 乙睛。3.2.2 甲醇。3.2.3 丙酣。3.2.4 乙酸乙醋。3.2.5 浓盐酸:优级纯。3.2.6 冰乙酸:优级纯。3.2.7 氨水:分析纯。3.2.8
4、盐酸:0.1mol/L，吸取浓盐酸0.83mL，用水定容于100mL容量瓶中。3.2.9 乙酸-乙酸乙醋(5+95):5 mL冰乙酸用乙酸乙醋定容至100mL。3.2. 10 氨水-乙睛(5+95):5mL氨水用乙腊定容至100mL。3. 2. 11 o. 45m滤膜。3.2. 12 7种硝基咪瞠标准品:纯度二三98.0%，其中罗硝略和二甲硝咪哩的代谢物为1-甲基-2-是甲基-5硝基咪嗤(简写为DMZOH)。3.2.13 标准储备液:分别称取每种硝基咪瞠标准品各10mg(准确至0.1mg)，用甲醇溶解，并分别定容到10mL棕色容量瓶中，混匀，该溶液的浓度为1mg/mL。避光一180C冷冻保存，
5、保存期为1年。3.2.14 混合标准中间液1:取每种标准储备液各1mL，移入100mL棕色容量瓶中，用甲醇定容。该溶液浓度为10g/mL。避光一180C冷冻保存，保存期为1年。3.2.15 t昆合标准中间液ll:取1mL混合标准中间液I于100mL棕色容量瓶中，用水稀释至刻度。该溶液浓度为100ng/mL。置于冰箱中lOC40C下避光保存，保存期为3个月。1 SN/T 1626一20053.2.16 标准工作液:根据需要，吸取一定量的混合标准中间液IT，用水稀释至所需浓度，现用现配。3.2.17 SCX固相萃取柱:5mL乙酸乙酸乙醋(5十95)预洗。空白实验有杂质干扰时，依次用10mL 氨水-
6、乙睛(5+95)、10mL O. 1 mol/L盐酸、20mL水和3mL甲醇、5mL乙酸-乙酸乙醋(5十95)预洗。整个过程应保持SCX柱湿润，最后柱上保留约1cm溶液，关闭活塞，柱上端连接储液器，贮液器底部塞一小块脱脂棉，备用。3.3 仪器和设备3.3. 1 高效液相色谱仪，配有紫外检测器。3.3.2 超声波清洗器。3.3.3 旋涡振荡器。3.3.4 旋转蒸发器。3.3.5 氮吹仪。3.3.6 均质器。3.3.7 离心机z最大转速5000 r/min。3.3.8 固相萃取装置。3.4 测定步骤3.4. 1 提取称取5g(准确至0.01g)样品于50mL离心管中，加入20mL乙酸乙醋，均质1m
7、in后，超声提取5 min，再以3000 r/min的速度离心10min，取上清液转移至鸡心瓶中。再用20mL乙酸乙醋重复提取一次，合并上清液于同一鸡心瓶中。3.4.2 净化将提取液于40.C下旋转蒸发至近干(不能全部蒸干)，用5mL乙酸-乙酸乙醋(5十95)溶解，将溶液转移到预洗过的SCX柱上的贮液器中，再用5mL乙酸-乙酸乙醋(5+95)洗鸡心瓶，洗液一并转移到SCX柱上的贮液器中，打开活塞，以2mL/min流速滴下。依次用2.5mL丙酣，5mL甲醇，5mL乙睛在2mL/min流速下淋洗柱子，用5mL氨水-乙睛(5+95)洗脱。弃去前2mL洗脱液，收集后面的洗脱液于玻璃管中，在40.C水活
8、上用氮气小心吹干，用水定容至1mL，游涡振荡或超声溶解，过0.45m滤液膜后，进行HPLC分析。3.4.3 测定3.4.3.1 灌相色i曾参考条件a) 色谱柱:C18柱，250mmX4.6 mm(内径)，填料颗粒直径5m或相当者。b) 流动相:A:水，B:乙睛，C:甲醇。洗脱程序见表1。时间/min。A/C%) 77 B/C%) 4 c/C%) 19 c) 动相流速:1. 0 mL/mino d) 紫外检测器波长:320 nmo u 色谱柱温度:350C。f) 进样量:50L。3.4.3.2 色谱测定表1方法的洗脱梯度程序5 12 77 71 4 10 19 19 L._ L_ 20 23 7
9、1 77 10 4 19 19 一-一一一28 77 4 19 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被测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，各被测物的保留时间见表2，标准品色谱图参见附录A。表2备被测物的保留时间被测物保留时间/min3.4.4 空白实验除不称取试样外，均按上述测定步骤进行。3.4.5 结果计算和表述用色谱数据处理软件中的外标法，或按式(1)计算试样中被测物的残留量:式中zX一(A- Ao) C. V -As.m X一一试样中被测物的残留量，单位为微克每千克(g/kg
10、);A一一样液被测物的色谱峰面积，单位为平方毫米(mm2); Ao -空白实验被测物的色谱峰面积，单位为平方毫米(mm2); 人标准工作液被测物的色谱峰面积，单位为平方毫米(mm2); C一一标准工作液中被测物的浓度，单位为纳克每毫升(ng/mL); V一一样液最终定容体积，单位为毫升(mL);m一一最终样液代表的试样量，单位为克(g)。4 测定低限、回收率4. 1 测定低限本方法的测定低限为:1g/kg。4.2 回收率SN/T 1626一2005( 1 ) 鸡肉在添加水平为lg/kg、2g/kg、5g/kg时，DMZOH的回收率在72.70%83. 86%之间;罗硝瞠的回收率在65.7%86
11、. 35%之间;甲硝略的回收率在69.85% 87. 14%之间;塞克硝哩的回收率在72.70% 83. 86 %之间;二甲硝眯哩的回收率在75.75% 91. 46%之间;替硝瞠的回收率在81. 00%90. 10%之间;奥硝瞠的回收率在92.73%1l7. 40%之间。猪肉在添加水平为1g/kg、2g/kg、5g/kg时，DMZOH的回收率在81.10 % 93. 50 %之间;罗硝瞠的回收率在62.20% 82. 40 %之间;甲硝瞠的回收率在76.68%88. 60%之间;塞克硝略的回收率在76.80% 88. 60 %之间;二甲硝咪哩的回收率在73.40% 97. 20%之间:替硝略
12、的回收率在93.50 % 99. 50 %之间;奥硝瞠的回收率在79.50%94. 00%之间。3 SNjT 1626-2005 0.8 0.7 0.6 0.5 0.4 3。b g 0.3 0.2 4 O. 1 。-0. 1 -0.2 。2 1一一-DMZOH;2一一-罗硝嗖43-一甲硝噬;4一一塞克硝唾p5一一二甲硝咪嗖$6一一替硝陪;7一一奥硝略。4 6 附录A(资料性附录)硝基咪睡标准晶液相色谱圄8 10 Minutes 12 图A.1硝基咪睡标准晶液相色谱圈14 16 18 20 SN/T 1626-2005 Foreword Annex A is an informative ann
13、ex. This standard was proposed band is under. the charge of National Regulatory Commission for Certi fication and Accreditation This standard was drafted by Hebei Entry-Exit Inspection and Ouarantine Bureau of the Peoples Re public of China. The main drafters of this standard are Wang Fengchi , Ma Z
14、hendong , Guo Chunhai , Lv Hongying and Ai Lianfeng This standard is an Entry-Exit Inspection and Ouarantine professional standard published for the first time. 5 SN/T 1626-2005 Determination of metronidazole, tinidazole, ornidazole, ronidazole ,dimetridazole and secnidazole residues in meat and mea
15、t products for import and export-HPLC 1 Scope The standard specifies the methods of determination of ronidazole (RNZ) , dimetridazole (DMZ) and their common metabolite-hdroxydimetridazole (DMZOH) , tinidazole (TNZ) , omidazole (ONZ) , secnidazole CSNZ) , and metronidazole (MNZ) , residues in meat an
16、d meat products bhigh-performance chromatography (HPLC). This standard is applicable to the determination of ronidazole (RNZ) , dimetridazole (DMZ) , and thei r common metabol ite-hydroxydimetridazole (DMZOH) , tinidazole (TNZ) , omidazole (ONZ) , secnidazole (SNZ) , and metronidazole (MNZ) residues
17、 in chicken and pork. 2 Sample preparations The laboratory samples are grinded by chopper. If the sample amount is too big , the representative parts should be taken from each piece of the sample and mixed well. The prepared sample are divid ed into two portions, one is ready to determine, and the o
18、ther one is sealed in a vessel and stored in dark at - 18 OC. 3 Method of determination 3. 1 Abstract of this method Nitroimidazoles residues in samples are extracted with ethyl acetate. The extraction is purified by SCX SPE column. The final solution are determined by HPLC with UV detector, using e
19、xternal stand ard method. 3. 2 Reagents and materials AII reagents used should be A. R. and water is deionized water, unless specifically noted. 3.2. 1 Acetonitrile. 3. 2. 2 Methanol. 6 SN/T 1626一20053. 2. 3 Acetone. 3.2.4 Eth1 acetate. 3. 2. 5 Hydrochloric acid: GR grade. 3. 2. 6 Acetic acid: GR gr
20、ade. 3. 2. 7 Ammonium hydroxide: analytical grade. 3.2.8 hydrochloric acid: O. 1 mol/L. Accurately suck O. 83 mL hydrochloric acid , dilut with deion ized water to scale in a 100 mL volumetric flask. 3. 2. 9 Acetic acid-eth1 acetate (5+95): Suck 5 mL acetic acid ,dilut with ethyl acetate to scale in
21、 a 100 mL volumetric flask. 3. 2. 10 Ammonium hydroxid-acetonitrile (5 + 95): Suck 5 mL ammonium hdroxide， dilut with ace tonitrile to scale in a 100 mL volumetric flask. 3.2.11 0.45m filter. 3.2.12 Standard of 7 nitroimidazoles: purity;?98.0%. 3.2. 13 Stock standard solution: Accurately weigh 10 mg
22、 ( accurate to O. 1 mg ) standard (3.2.12) ,dissolve with methanol to scale in a 20 mL umber volumetric flask individuall. The con centration of the solutions are 1 mg/mL. The solutions should be stored at一18oC in dark and keep stable for one year. 3. 2. 14 Mixed medium standard solution 1 : Suck 1.
23、 00 mL stock standard solution of 7 nitroimid azoles (3.2. 13) respectively into a 100 mL umber volumetric flask , dilut with methenol to the scale and mix well. The concentration of the solution is 10.0g/mL. The solutions should be stored at - 18 oC in dark and keep stable for one year. 3.2.15 Medi
24、um Standard solution n : Suck 1.00 mL mixed standard medium solution 1(3.2. 14) in to a 100 mL umber volumetric flask , dilut with methenolto the scale and mix well. The concentration of solution is 100 ng/mL. The solutions should be stored at 10C _40C in dark and keep stable for three months. 3.2.1
25、6 Working standard solution: Accoiding to the concentration required , with deionized water dilut medium standard solution n (3. 2. 15) to working standard solution , use immidiately after preparation. 7 SN/T 1626-2005 3.2.17 SCX solid-phase extracrion cartridge: 5 mL acetic acid-ethyl acetate (5 +
26、95) pass the臼rtridge to condition. If there is interference in blank test, the cartridge should be washed as follows: 10 mL ammonium hydroxide-acetonitrile (5 + 9酌，0.1 mol/L hdrochloric acid , 20 mL deionized wa ter, 3 mL methenol and 5 mL acetic acid-ethyl acetate (5 + 95). In this process , the SC
27、X cartridge should keep wet and leave about 1 cm high solution in cartridge. Then connect a reservoi门heOOt tom of which is stuffed with degrease cotton to the top of cartridge. 3. 3 Apparatus and equipment 3.3. 1 High-performance chromatograph: equipment with UV detector. 3. 3. 2 Ultrasonic cleanser
28、. 3.3.3 Vortex shaker. 3.3.4 Rotary vacuum evaparator. 3. 3. 5 Nitrogen evaparator. 3. 3. 6 Homogenizer. 3.3.7 Centrifuge: the max rotate speed is 5000 r/min. 3.3.8 Vacuum manifold processing station. 3. 4 Procedure 3. 4. 1 Extraction Weigh ca 5 9 of test sample (accurate to O. 01 g) into a 50 mL ce
29、ntrifuge tube, add 20 mL eth1 ace ta饵，homogenize 1 min , extract for 5 min in ultrasonic bath and centrifuge for 5 min at 3 000 r/min. Transfer the extraction into the heart-shape flask and extract once more with another 20 mL ethyl ac etate and combine the extraction into the臼meflask. 3. 4. 2 Clean
30、-up Evaporate the exaction to almost dryness (not complete dryness) using rotary vacuum evaparator at 40 .C. Dissovle the residues with 5 mL acetic acid-ethI acetate(5 + 95) , transfer the solution into the reservoir above the conditioned SCX cartridge. Dissolve the residues again with 5 ml acetic a
31、cid-ethyl acetate(5+95) and pour into the same reservoir. Then let the solution pass the cartridge at the speed of 2 mL/min. After drop over, wash the cartridge with 2. 5 mL acetone. 5 mL methan ole , 5 mL acetontrile in turn , finally elute the cartridge with 5 mL ammonium hydroxide-acetonitrile (5
32、 + 95) , discard the prime 2 mL elution and collect the left elution with 10 mL glass flask. Evaporate elution to dryness with nitrogen evaporator at 40 C. Residues are dissolved with 1.0 ml deionized water in ultrasonic bath and mix well by vortex shaker. Then the solution is passed through O. 45m
33、8 SN/T 1626-2005 filter and readfor HPLC analsis. 3. 4. 3 Determination 3.4.3.1 HPLC operating conditions a) Column: C18 150 mm x 4. 6 mm (i. d. ) 5m particle size. b) Mobile phase: A: deionized water 8: acetontrile C: methanole. The gradient program was showed in table 1. Table 1 The gradient progr
34、am Time/min 。5 12 20 23 28 A/%) 77 n 71 71 77 77 8/(%) 4 4 10 10 4 4 C/(%) 19 19 19 19 19 19 c) Flow rate: 1.0 mL/min. d) UV Detector: 320 nm. e) Column temperature: 35 oC. f) Sample volume: 50L. 3. 4. 3. 2 HPLC determination According to the approximate concentration of nitroimidazoles residues in
35、sample. select the stand ard working solution with similar responses to that of sample solution. The reponses of nitroimid azoles in the working standard and the sample solution should be within the linear range of the instru me时.he working standard solution and the sample solution should be injecte
36、d alternatively. Under the above HPLC operating condition. the retention time of nitroimidazoles seeing table 2. The chro matogram of the standard solution is shown in annex A. Table 2 Rentention time of seven nitroimidazoles Analytes I DMZOH I RNZ I MNZ I SNZ ! DNZ I TNZ I ONZ RententionTime I 7.01
37、 I 7.46 I 7.87 I 10.84 I 11.31 ! 12.91 I 18.07 3. 4. 4 Blank 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. 3. 4. 5 Calculation an expression of result Calculation the content of nitroimidazoles residues in t
38、est sample by HPLC data processor or accord-9 SN/T 1626一2005ing to formula (1) x = (A - Ao) c V As. m 、，E J，、. . . . . . . . . . . . . . . . . . . . . . . . . . . . . where: X一一theresidue of ntromdazoles n test sample ,mg/kg; A -the peak area of analyte of the sample soluton , mm勺A。一一-thepeak area o
39、f blank test, mm勺As一一-thepeak area of analytes of the standard soluton , mm勺c一一theconcentraton of nitroimidazoles in standard solution，g/mL; V一-thefnal volume of the sample solution, mL; m一-thecorresponding mass of test sample in the final solution , g. 4 Limit of determination and recovery 4. 1 Lim
40、it of determination The limit of determination of this method is 1g/kg. 4. 2 Recovery The recovery range of DMOH is 72. 70% to 83. 86%; the recovery range of RMZ is 65. 7% to 86. 35 % ; the recovery range of AMZ is 69. 85 % to 87. 14%; the recovery range of SNZ is 72. 70 % to 83.86%; the recovery ra
41、nge of DMZ is 75. 75% to 91. 46%; the recove叩rangeof TNZ is 81. 00% to 90. 10%; the recovery range of ONZ is 92. 73% to 117.40% , when fortified at the concentration of 0.1 mg/kg, 0.5 mg/kg and 1.0 mg/kg. The recove叩rangeof DMOH s 81. 10% to 93.50%; the recovery range of RMZ is 62.20% to 82.40%; the
42、 recove叩rangeof AMZ is 76. 68% to 89.60%; the recovery range of SNZ is 76. 80% to 88. 60%; the recoverrange of DMZ is 73. 40% to 97.20%; the recovery range of TNZ is 93. 50% to 99. 50%; the recovery range of ONZ is 79. 503% to 94. 00% , when fortified at the concentration of 0.1 mgl闸，0.5 mg/kg and 1
43、.0 mg/kg. 10 0.8 0.7 0.6 0.5 0.4 3。=E , 0.3 0.2 O. 1 0.0 O. 1 自0.2。1一一-DMZOH;2一一-RNZ;3一一-MNZ;4一一-SNZ;5-DNZ; 6-TNZ; 7一一一ONZ.2 4 Annex A (informative annex) Chromatogram of the standards 6 8 10 Minutes 、.t)12 14 16 Fig. A. 1 the chromatogram of senven nitroimidazoles standards SN/T 1626-2005 18 20 CON-创-HZ中华人民共和国出入境检验检疫行业标准进出口肉及肉制晶中甲硝砸、替硝睡、奥硝睡、罗硝睡、二甲硝眯睡、塞克硝睡残留量测定方法高效液相色谱法SN/T 1626-2005 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l字数22千字2005年11月第一次印刷开本880X 1230 1/16 2005年11月第一版 7G 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)68533533定价10.00书号，155066.2-16470 SN/T 1626-2005
