高效液相色谱-串联质谱法测定大鼠组织中的吡蚜酮
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摘要
为研究不同染毒剂量下吡蚜酮原药在大鼠体内的毒代动力学特征,以SD大鼠为研究对象,经口灌胃方式单次染毒,采集不同时间点的动物组织样品,经蛋白沉淀处理后,以吡虫啉为内标,采用高效液相色谱-串联质谱法(HPLC-MS/MS)检测大鼠组织中的吡蚜酮原型。结果表明,吡蚜酮在50～10 000μg/L浓度范围内,线性关系良好,相关系数大于0.98,批内精密度相对标准偏差小于15%。经口染毒后,吡蚜酮在大鼠体内分布较广,给药10 min后,在主要脏器中均检测到吡蚜酮原型,2 h后在多数组织中吡蚜酮浓度达最高峰,而至144 h,除肝脏外,其他组织中吡蚜酮浓度均降至最低定量限以下。此结果与血浆动力学结果吻合,表明该药物在大鼠体内不会长时间蓄积,安全性较高。
In order to study the toxicokinetic characteristics of pyridoxine in rats at different doses, SD rats were used as the research object, and single-time exposure was carried out by oral gavage. Animal tissue samples were collected at different time points. After the samples were treated with protein precipitation, the pyridoxine prototype was detected by high performance liquid chromatograpy-tandem mass spectrometry(HPLC-MS/MS) method with imidacloprid as internal standard. The results showed that the linear relationship of pymetrozine is good in the concentration range of 50-10 000 μg/L with the relative coefficient more than 0.98, RSD of intra-assay precision is less than 15%. After oral administration, pymetrozine was distributed in all organs of rats. At 10 min, the drug concentration in the lung was the highest, followed by spleen, heart, muscle and brain. Compare the changes of drug concentration in different organ tissues over time, it can be found that the peak time of drug concentration in brain and muscle appeared at 10 min, while in other tissues appeared at 2 h. The peaks were sorted in descending order, and the results were: liver>spleen>heart>lung>kidney>muscle>brain>testis>fat. After the drug concentration peaked, the drug concentration in each organ tissue gradually decreased with time. By 144 h, no pymeidone could be detected in all organs except for a small amount of pymetrozine. This result is consistent with the plasma kinetics results, which proves that the pesticide does not accumulate in rats for a long time and has high safety. The above results indicate that pymetrozine can be widely distributed in various organs of rats, and its dynamic distribution in tissues is basically consistent with the trend in plasma, and the elimination is faster, no accumulation tendency. At the same time, pymetrozine can quickly reach the brain tissue through the blood-brain barrier, or enter the testicular tissue through the blood testis barrier, but by 144 h, it is eliminated to below the minimum detection limit, and it has no teratogenic effects. These results provide a basis for the chronic toxicity of pymetrozine and the dose selection for carcinogenicity studies, and provide data support for its safety evaluation and methodological references for subsequent researchers. In this study only the distribution of pyridoxine prototype in rats was examined, and its metabolites was not able to be detected. It is necessary to develop more precise methods to conduct in deep studies to clarify distribution and excretion of metabolites in animals.
In order to study the toxicokinetic characteristics of pyridoxine in rats at different doses, SD rats were used as the research object, and single-time exposure was carried out by oral gavage. Animal tissue samples were collected at different time points. After the samples were treated with protein precipitation, the pyridoxine prototype was detected by high performance liquid chromatograpy-tandem mass spectrometry(HPLC-MS/MS) method with imidacloprid as internal standard. The results showed that the linear relationship of pymetrozine is good in the concentration range of 50-10 000 μg/L with the relative coefficient more than 0.98, RSD of intra-assay precision is less than 15%. After oral administration, pymetrozine was distributed in all organs of rats. At 10 min, the drug concentration in the lung was the highest, followed by spleen, heart, muscle and brain. Compare the changes of drug concentration in different organ tissues over time, it can be found that the peak time of drug concentration in brain and muscle appeared at 10 min, while in other tissues appeared at 2 h. The peaks were sorted in descending order, and the results were: liver>spleen>heart>lung>kidney>muscle>brain>testis>fat. After the drug concentration peaked, the drug concentration in each organ tissue gradually decreased with time. By 144 h, no pymeidone could be detected in all organs except for a small amount of pymetrozine. This result is consistent with the plasma kinetics results, which proves that the pesticide does not accumulate in rats for a long time and has high safety. The above results indicate that pymetrozine can be widely distributed in various organs of rats, and its dynamic distribution in tissues is basically consistent with the trend in plasma, and the elimination is faster, no accumulation tendency. At the same time, pymetrozine can quickly reach the brain tissue through the blood-brain barrier, or enter the testicular tissue through the blood testis barrier, but by 144 h, it is eliminated to below the minimum detection limit, and it has no teratogenic effects. These results provide a basis for the chronic toxicity of pymetrozine and the dose selection for carcinogenicity studies, and provide data support for its safety evaluation and methodological references for subsequent researchers. In this study only the distribution of pyridoxine prototype in rats was examined, and its metabolites was not able to be detected. It is necessary to develop more precise methods to conduct in deep studies to clarify distribution and excretion of metabolites in animals.
引文
[1] 冯义志,潘金菊,刘伟.超高效液相色谱质谱联用检测小麦中吡蚜酮的残留分析方法[J].农药,2015,54(1):48-50.FENG Yizhi,PAN Jinju,LIU wei.Analysis of pymetrozine residue in wheat by UPLC-MS/MS[J].Agrochemicals,2015,54(1):48-50(in Chinese).
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[3] 刘建华,张小峰,季美娟,刘文华.吡蚜酮原药高效液相色谱分析[J].农药,2008,47(10):741-742.LIU Jianhua,ZHANG Xiaofeng,JI Meijuan,LIU Wenhua.A HPLC analysis method for pymetrozine technical[J].Agrochemicals,2008,47(10):741-742(in Chinese).
[4] 吴亚坚,徐赛,李飞,韩冲冲,彭文文,李保同.0.5%吡蚜酮·杀虫双缓释粒的 HPLC 分析[J].现代农药,2018,17(1):29-31.WU Yajian,XU Sai,LI Fei,HAN Chongchong,PENG Wenwen,LI Baotong.Analysis of pymetrozine+bisultap 0.5% BRG by HPLC[J].Modern Agrochemicals,2018,17(1):29-31(in Chinese).
[5] 齐畅,刘美欧,杨珊珊,常潇晨,李月秋,张爱霞.分散液相微萃取荧光法测定果蔬中残留吡蚜酮[J].农药,2017,56(6):434-436.QI Chang,LIU Meiou,YANG Shanshan,CHANG Xiaochen,LI Yueqiu,ZHANG Aixia.Determination of pymetrozine residues in fruits and vegetables by dispersive liquid-liquid microextraction and fluorescence spectrophotometry[J].Agrochemicals,2017,56(6):434-436(in Chinese).
[6] 于巍,林立红,王刚,孙常松,孟庆贺,李晓磊,段明郁.吡蚜酮在大鼠体内的毒代动力学研究[J].化学与生物工程,2014,31(7):53-56.YU Wei,LIN Lihong,WANG Gang,SUN Changsong,MENG Qinghe,LI Xiaolei,DUAN Mingyu.Study on toxicokinetics of pymetrozine in rat[J].Chemistry&Bioengineer,2014,31(7):53-56(in Chinese).
[7] 农业部农药检定所.GB 15670—1995 《农药登记毒理学实验方法》[S].北京:中国标准出版社,1995.
[8] 农业部农药检定所.经济合作与发展组织化学品测试准则[M].北京:中国农业出版社,2012.
[9] 国家药典委员会.《中华人民共和国药典》第四部[M].北京:中国医药科技出版社,2015:363.
[10] LIN L H,YU W,PANG Y N,SUN C S.Validation of an LC-MS/MS method for the quantitative determination of a novel fungicide:pyraoxystrobin in rat plasma and its application to a toxicokinetics study[J].Anal Bioanal Chem,2014(406):5 521-5 526.
[11] 王恩鹏,斋藤哲男,孙岩,陈长宝,刘淑莹.高分离快速液相色谱-四极杆飞行时间质谱法检测人参皂苷Re在大鼠体内的分布[J].质谱学报,2016,37(6):526-532.WANG Enpeng,SAITO Tetsuo,SUN Yan,CHEN Changbao,LIU Shuying.Tissue distribution of ginsenoside Re in rats by rapid resolution liquid chromatography coupled with quadrup[J].Journal of Chinese Mass Spectrometry Society,2016,37(6):526-532(in Chinese).
[12] 刘海培,孟繁华,郭继芬,司端运,朱晓薇,赵毅民.HPLC-MS/MS法分析大鼠体内淫羊藿素葡糖醛酸结合物[J].质谱学报,2010,31(6):376-379.LIU Haipei,MENG Fanhua,GUO Jifen,SI Duanyun,ZHU Xiaowei,ZHAO Yimin.Analysis of glucuronide conjugates of icaritin in rat by LC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2010,31(6):376-379(in Chinese).
[13] 杨光,高明伟,严婉静,李增刚,吴英良.吡蚜酮原药大鼠致畸毒性实验[J].农药,2014,53(10):753-755.YANG Guang,GAO Mingwei,YAN Wanjing,LI Zenggang,WU Yingliang.Teratogenicity test of pymetrozine TC in rats[J].Agrochemicals,2014,53(10):753-755(in Chinese).
[2] 徐金丽,徐真真,徐光军,刘修园,周杨全,王秀国.吡蚜酮在烟草中的残留降解及风险评估[J].中国烟草科学,2015,36(5):69-73.XU Jinli,XU Zhenzhen,XU Guangjun,LIU Xiuyuan,ZHOU Yangquan,WANG Xiuguo.Dissipation and residual risk of pymetrozine in tobacco[J].Chinese Tobacco Science,2015,36(5):69-73(in Chinese).
[3] 刘建华,张小峰,季美娟,刘文华.吡蚜酮原药高效液相色谱分析[J].农药,2008,47(10):741-742.LIU Jianhua,ZHANG Xiaofeng,JI Meijuan,LIU Wenhua.A HPLC analysis method for pymetrozine technical[J].Agrochemicals,2008,47(10):741-742(in Chinese).
[4] 吴亚坚,徐赛,李飞,韩冲冲,彭文文,李保同.0.5%吡蚜酮·杀虫双缓释粒的 HPLC 分析[J].现代农药,2018,17(1):29-31.WU Yajian,XU Sai,LI Fei,HAN Chongchong,PENG Wenwen,LI Baotong.Analysis of pymetrozine+bisultap 0.5% BRG by HPLC[J].Modern Agrochemicals,2018,17(1):29-31(in Chinese).
[5] 齐畅,刘美欧,杨珊珊,常潇晨,李月秋,张爱霞.分散液相微萃取荧光法测定果蔬中残留吡蚜酮[J].农药,2017,56(6):434-436.QI Chang,LIU Meiou,YANG Shanshan,CHANG Xiaochen,LI Yueqiu,ZHANG Aixia.Determination of pymetrozine residues in fruits and vegetables by dispersive liquid-liquid microextraction and fluorescence spectrophotometry[J].Agrochemicals,2017,56(6):434-436(in Chinese).
[6] 于巍,林立红,王刚,孙常松,孟庆贺,李晓磊,段明郁.吡蚜酮在大鼠体内的毒代动力学研究[J].化学与生物工程,2014,31(7):53-56.YU Wei,LIN Lihong,WANG Gang,SUN Changsong,MENG Qinghe,LI Xiaolei,DUAN Mingyu.Study on toxicokinetics of pymetrozine in rat[J].Chemistry&Bioengineer,2014,31(7):53-56(in Chinese).
[7] 农业部农药检定所.GB 15670—1995 《农药登记毒理学实验方法》[S].北京:中国标准出版社,1995.
[8] 农业部农药检定所.经济合作与发展组织化学品测试准则[M].北京:中国农业出版社,2012.
[9] 国家药典委员会.《中华人民共和国药典》第四部[M].北京:中国医药科技出版社,2015:363.
[10] LIN L H,YU W,PANG Y N,SUN C S.Validation of an LC-MS/MS method for the quantitative determination of a novel fungicide:pyraoxystrobin in rat plasma and its application to a toxicokinetics study[J].Anal Bioanal Chem,2014(406):5 521-5 526.
[11] 王恩鹏,斋藤哲男,孙岩,陈长宝,刘淑莹.高分离快速液相色谱-四极杆飞行时间质谱法检测人参皂苷Re在大鼠体内的分布[J].质谱学报,2016,37(6):526-532.WANG Enpeng,SAITO Tetsuo,SUN Yan,CHEN Changbao,LIU Shuying.Tissue distribution of ginsenoside Re in rats by rapid resolution liquid chromatography coupled with quadrup[J].Journal of Chinese Mass Spectrometry Society,2016,37(6):526-532(in Chinese).
[12] 刘海培,孟繁华,郭继芬,司端运,朱晓薇,赵毅民.HPLC-MS/MS法分析大鼠体内淫羊藿素葡糖醛酸结合物[J].质谱学报,2010,31(6):376-379.LIU Haipei,MENG Fanhua,GUO Jifen,SI Duanyun,ZHU Xiaowei,ZHAO Yimin.Analysis of glucuronide conjugates of icaritin in rat by LC-MS/MS[J].Journal of Chinese Mass Spectrometry Society,2010,31(6):376-379(in Chinese).
[13] 杨光,高明伟,严婉静,李增刚,吴英良.吡蚜酮原药大鼠致畸毒性实验[J].农药,2014,53(10):753-755.YANG Guang,GAO Mingwei,YAN Wanjing,LI Zenggang,WU Yingliang.Teratogenicity test of pymetrozine TC in rats[J].Agrochemicals,2014,53(10):753-755(in Chinese).
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