液相色谱-串联质谱法研究氟噻草胺在大鼠体内组织中的分布行为
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摘要
建立了大鼠组织中氟噻草胺的液相色谱-串联质谱(LC-MS/MS)分析方法,并对其经灌胃给药后在大鼠体内各组织中的分布行为进行了研究。大鼠组织样品经乙腈提取后,采用LCMS/MS法测定。色谱柱为Phenomenex反向C_(18)色谱柱(50 mm×4.6 mm,5μm),以体积分数为72%乙腈水溶液(含体积分数为0.1%甲酸)为流动相,等度洗脱,流速0.6 mL/min。质谱采用电喷雾离子源(ESI)及多重反应监测(MRM)模式,定量离子对为m/z 364.1/194.2。大鼠心、肝、脾、肺、肾、脑、肌肉、睾丸和脂肪在0.002～0.1 mg/L范围内,大肠、小肠和胃在0.10～10 mg/L范围内,氟噻草胺的质量浓度与对应的峰面积间线性关系良好(r> 0.99)。质控样品的相对标准偏差均小于11%,基质效应在94.5%～95.6%之间,提取回收率在96%～107%之间,稳定性在95%～104%之间,均符合方法学要求。经单次灌胃给予氟噻草胺400 mg/kg后,其主要分布于大鼠的胃、大肠、小肠和肺中,各组织浓度达峰值后,随着时间延长均可从体内消除。表明氟噻草胺在动物体内分布广泛且降解迅速。本研究建立了一种可用于测定大鼠组织中氟噻草胺浓度的高效、专属、灵敏的LC-MS/MS方法,进而阐明了氟噻草胺的组织分布行为。
A method using liquid chromatography-tandem mass spectrometry(LC-MS/MS) was developed for the determination of flufenacet in rat tissues and the investigation of the tissues distribution behavior of test item in rats after intragastric administration. Rat tissue homogenate was extracted withacetonitrile and analyzed by LC-MS/MS. Chromatographic separation was performed on a Phenomenex reverse C_(18)(50 mm × 4.6 mm, 5 μm) column at a flow rate of 0.6 mL/min with iscratic elution. The mobile phase was 72% acetonitrile/water solution(containing 0.1% formic acid).Electrospray ionization(ESI) with a positive-ion and multiple reaction monitoring mode was used.Quantitative ion pair was 364.1/194.2. The linear ranges of flufenacet in heart, liver, spleen, lung,kidney, brain, muscle, testis and fat were all ranged from 0.002 to 0.1 mg/L. The linear ranges of flufenacet in large intestine, small intestine and stomach were in the range of 0.10-10 mg/L. The correlation coefficient between the mass concentration of flufenacet and the corresponding peak area was good(r > 0.99). The precision of this method for QC samples was less than 11%. The matrix effect was in the range of 94.5%-95.6%. The recovery was in 96 %-107%, and the stability was in 95%-104%.All these parameters met the requirements of methodology. The experimental results of tissue distribution of flufenacet showed that flufenacet was widely and rapidly distributed in rats after the gavage of 400 mg/kg flufenacet. Flufenacet mainly distributed in large intestine, small intestine,stomach and lung. And after the concentration of each tissue reached the peak, flufenacet could be eliminated in all tissues. An efficient, specific and sensitive LC-MS/MS method was established, and this validated method could be used in the analysis of flufenacet in rat tissues to realize the tissue distribution behaviors.
A method using liquid chromatography-tandem mass spectrometry(LC-MS/MS) was developed for the determination of flufenacet in rat tissues and the investigation of the tissues distribution behavior of test item in rats after intragastric administration. Rat tissue homogenate was extracted withacetonitrile and analyzed by LC-MS/MS. Chromatographic separation was performed on a Phenomenex reverse C_(18)(50 mm × 4.6 mm, 5 μm) column at a flow rate of 0.6 mL/min with iscratic elution. The mobile phase was 72% acetonitrile/water solution(containing 0.1% formic acid).Electrospray ionization(ESI) with a positive-ion and multiple reaction monitoring mode was used.Quantitative ion pair was 364.1/194.2. The linear ranges of flufenacet in heart, liver, spleen, lung,kidney, brain, muscle, testis and fat were all ranged from 0.002 to 0.1 mg/L. The linear ranges of flufenacet in large intestine, small intestine and stomach were in the range of 0.10-10 mg/L. The correlation coefficient between the mass concentration of flufenacet and the corresponding peak area was good(r > 0.99). The precision of this method for QC samples was less than 11%. The matrix effect was in the range of 94.5%-95.6%. The recovery was in 96 %-107%, and the stability was in 95%-104%.All these parameters met the requirements of methodology. The experimental results of tissue distribution of flufenacet showed that flufenacet was widely and rapidly distributed in rats after the gavage of 400 mg/kg flufenacet. Flufenacet mainly distributed in large intestine, small intestine,stomach and lung. And after the concentration of each tissue reached the peak, flufenacet could be eliminated in all tissues. An efficient, specific and sensitive LC-MS/MS method was established, and this validated method could be used in the analysis of flufenacet in rat tissues to realize the tissue distribution behaviors.
引文
[1]European Commission.Review report for the active substance flufenacet.[EB/OL].(2003-07-03).http://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=activesubstance.detail&languag=EN&selectedID=1380.
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[3]黎育生,周桂东,沈宜全,等.氟噻草胺的合成[J].现代农药,2002,1(2):8-10.LI Y S,ZHOU G D,SHEN Y Q,et al.Synthesis of flufenacet[J].Mod Agrochem,2002,1(2):8-10.
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[5]SOLTANI N,DEEN B,BOWLEY S,et al.Effects of pre-emergence applications of flufenacet plus metribuzin on weeds and soybean{Glycine max)[J].Crop Prot,2005,24(6):507-511.
[6]KLEEMANN S G L,BOUTSALIS P,GILL G S,et al.Applications of pre-emergent pyroxasulfone,flufenacet and their mixtures with triallate for the control of Bromus dia,ndrus(ripgut brome)in no-till wheat(Triticum aestivum)crops of southern Australia[J].Crop Prot,2016,80:144-148.
[7]BAZOOBANDI M,YADURAJU N T,KULSHRESTHA G.Analysis of flufenacet in soil,wheat grain and straw by gas chromatography[J].J Chromatogr A,2000,886(1-2):319-322.
[8]WANG D H,ZHANG Q,ZHENG Y,et al.Estimating the combined toxicity of flufenacet and imazaquin to Sorghum with pore water herbicide concentration[J].J Environ Sci,2016,41:154-161.
[9]农药登记毒理学试验方法:GB15670—2017[S].北京:中国农业出版社,2017.Toxicological test methods of pesticides for registration:GB15670—2017[S].Beijing:China Agricultural Press,2017.
[10]生物样品定量分析方法验证指导原则:中华人民共和国药典2015年版.四部[M].北京:中国医药科技出版社,2015:363.Guidance on bioanalysis:Method validation and analysis of study samples:The pharmacopoeia of the People's Republic of China 2015Edition.VolⅤ[M].Beijing:China Medical Science Press,2015:363.
[2]刘宝擅.氟噻草胺的合成研究[D].石家庄:河北科技大学,2013.LIU B S.Study on the synthesis process of flufenacet[D].Shijiazhuang:Hebei University of Science and Technology,2013.
[3]黎育生,周桂东,沈宜全,等.氟噻草胺的合成[J].现代农药,2002,1(2):8-10.LI Y S,ZHOU G D,SHEN Y Q,et al.Synthesis of flufenacet[J].Mod Agrochem,2002,1(2):8-10.
[4]陈杰,沈建.氟噻草胺原药的高效液相色谱分析[J].农药科学与管理,2010,31(9):46-48.CHEN J,SHEN J.Analysis of flufenacet by HPLC[J].Pest Sci Admin,2010,31(9):46-48.
[5]SOLTANI N,DEEN B,BOWLEY S,et al.Effects of pre-emergence applications of flufenacet plus metribuzin on weeds and soybean{Glycine max)[J].Crop Prot,2005,24(6):507-511.
[6]KLEEMANN S G L,BOUTSALIS P,GILL G S,et al.Applications of pre-emergent pyroxasulfone,flufenacet and their mixtures with triallate for the control of Bromus dia,ndrus(ripgut brome)in no-till wheat(Triticum aestivum)crops of southern Australia[J].Crop Prot,2016,80:144-148.
[7]BAZOOBANDI M,YADURAJU N T,KULSHRESTHA G.Analysis of flufenacet in soil,wheat grain and straw by gas chromatography[J].J Chromatogr A,2000,886(1-2):319-322.
[8]WANG D H,ZHANG Q,ZHENG Y,et al.Estimating the combined toxicity of flufenacet and imazaquin to Sorghum with pore water herbicide concentration[J].J Environ Sci,2016,41:154-161.
[9]农药登记毒理学试验方法:GB15670—2017[S].北京:中国农业出版社,2017.Toxicological test methods of pesticides for registration:GB15670—2017[S].Beijing:China Agricultural Press,2017.
[10]生物样品定量分析方法验证指导原则:中华人民共和国药典2015年版.四部[M].北京:中国医药科技出版社,2015:363.Guidance on bioanalysis:Method validation and analysis of study samples:The pharmacopoeia of the People's Republic of China 2015Edition.VolⅤ[M].Beijing:China Medical Science Press,2015:363.