液相微萃取—高效液相色谱法在中药分析中的应用

【作者】 杨雪；

【导师】 白小红；

【作者基本信息】 山西医科大学 ， 药物分析， 2007， 硕士

【摘要】 目的建立单滴液相微萃取-高效液相色谱法测定槐花米及其制剂血栓心脉宁胶囊中芦丁含量的方法。方法利用自制液相微萃取装置,选用100μl的正丁醇为萃取有机溶剂、0.01 mol/L HCl3.00 ml为供相、搅拌速度为600 r/min和萃取时间为10 min,萃取后,用100μl甲醇溶解萃出物,进样20μl经高效液相色谱仪分析,采用C＿（18）柱,流动相为甲醇:1%冰醋酸（50:50）,流速0.8ml/min,柱温30℃,在波长257 nm处,测定中药及其制剂中的芦丁含量。结果芦丁的线性范围为10～100μg/ml（n=5,r=0.9925）,槐花米中芦丁的平均加样回收率为（101.7±5.5）%（n=9）,血栓心脉宁胶囊中芦丁的平均加样回收率为（103.44±3.3）%（n=9）,RSD均小于8.5%,检测限1.0μg/ml。结论该方法能有效地去除复杂基体的干扰,有机溶剂消耗少,作为一种快速、有效、灵敏的复杂样品前处理技术,可用于槐花米及其制剂血栓心脉宁胶囊中芦丁的含量测定。目的建立基于中空纤维为支持体的液相微萃取/后萃取-高效液相色谱法同时测定中药苦参、复方苦参注射液及生物样品中氧化苦参碱和苦参碱含量的方法。方法利用自制的微萃取装置,选择异丙醇为萃取有机溶剂,pH9的NaOH 2.00 ml为供相,pH4的HCl为接受相,聚丙烯腈纤维的长度为10 cm,搅拌速度为1500 r/min,萃取时间为30min。萃取完成后,进样20μl经高效液相色谱仪分析,采用C₁₈柱,流动相为甲醇:pH6.0磷酸盐缓冲液（57:43）,流速为0.8 ml/min,柱温35℃,在波长220 nm处,测定中药、制剂及生物样品中氧化苦参碱和苦参碱的含量。结果中药及制剂中,线性范围为:氧化苦参碱11～437μg/ml,苦参碱10～433μg/ml,相关系数均大于0.999。精密度实验:氧化苦参碱RSD均小于9.4%,苦参碱RSD均小于6.7%。复方苦参注射液中平均回收率:氧化苦参碱83.0%～116.1%;苦参碱108.8%～117.8%。苦参药材中氧化苦参碱的平均回收率为:104.3%～114.7%。检测限为:氧化苦参碱1.0μg/ml;苦参碱1.0μg/ml。血浆中线性范围为:氧化苦参碱5～119μg/ml,苦参碱3～117μg/ml,相关系数均大于0.997。精密度实验:氧化苦参碱RSD均小于5.9%,苦参碱RSD均小于5.1%。平均回收率:氧化苦参碱88.8%～106.4%;苦参碱96.0%～104.2%。检测限为:氧化苦参碱1.4μg/ml;苦参碱1.0μg/ml。肝脏中线性范围为:苦参碱3～131μg/ml（n=5,r=0.9995）。精密度实验:苦参碱RSD均小于6.8%。苦参碱平均回收率为:93.7%～101.6%。苦参碱检测限为:1.0μg/ml。结论该方法能有效去除复杂机体的干扰,提高了选择性,有机溶剂消耗少,是一种快速、精确、灵敏的同时测定氧化苦参碱和苦参碱的方法。目的基于中空纤维支持的非水液相微萃取/后萃取-高效液相色谱法同时测定中药银杏叶中槲皮素、山柰素和异鼠李素的含量。方法利用自制的微萃取装置,考察了中空纤维种类、萃取溶剂种类、供相与接受相pH、搅拌速度、萃取时间的影响,选定了聚砜纤维长度为10 cm,萃取溶剂为正辛醇,接受相为pH10的NaOH,供相为2.00 ml含HCl10^-3mol/L的甲醇溶液,搅拌速度为1500 r/min,萃取时间为30 min。萃取完成后,进样20μl经高效液相色谱仪分析,采用C₁₈柱,流动相为甲醇:0.4%磷酸（60∶40）,流速为0.8 ml/min,柱温30℃,在波长360 nm处分析测定,在该条件下达到了令人满意的萃取效果。结果槲皮素、山柰素与异鼠李素的线性范围分别为:3.63～49.36μg/ml、3.63～49.36μg/ml和1.85～25.19μg/ml,相关系数均大于0.994。平均回收率分别为:（95.6±4.8）%（n=5）、（107.5±5.3）%（n=5）和（103.4±4.1）%（n=5）。RSD分别为:4.6%、3.7%和4.3%。检测限分别为:40、70和90 ng/ml。结论本文首次提出非水液相微萃取/后萃取方式,并成功地将其应用于中药中槲皮素、山柰素和异鼠李素的含量测定。更多还原

【Abstract】 Objective Single-drop liquid-phase microextraction（SD-LPME）coupled with high performance liquid chromatography（HPLC）was developed for the determination of rutin in Sophora japonica L.and capsula preparation.Method A self-made LPME system was adopted.The optimal experimental conditions,100μl n-butanol as organic solvent,3.00 ml 0.01 mol/L HCl as the donor aqueous solution,600 r/min of the stirring rate,and 10 min of the extraction time,were selected.After extraction,the extractant was dissolved in 100μl methanol.An aliquot（20μl）was injected into an HPLC system with methanol:1%HAc（50∶50）as the mobile phase.The C₁₈column was used with flow rate at 0.8 ml/min and temperature at 30℃.The detective wave length was set at 257 nm.Under the optimal conditions high extract efficiency was obtained.Result The linear range of rutin is from 10 to 100μg/ml with the correlation coefficient（r） 0.9925.The average relative recoveries of rutin in Sophora japonica L.and capsula were （101.7±5.5）%（n=9）and（103.4±3.3）%（n=9）respectively.The relative standard deviation was lower than 8.5%.The limit of detection was 1.0μg/ml for rutin.Conclusion This method was applied to eliminate the interference of matrices,and to decrease the volume of use of organic solvent,and results showed the possibility for the determination of rutin in Sophora japonica L.and capsula preparation.It is a quick,efficient and sensitive pre-processing technique. Objective Liquid-phase microextraction with back extraction（LPME/BE）based on porous hollow fibres coupled with high performance liquid chromatography（HPLC）was developed for the simultaneous determination of oxymatrine and matrine in Sophora flavescens Ait.,injection preparation and biological matrices.Method The effects of kinds of hollow fiber and microextraction solvent,pH in the donor and acceptor phases,stirring rate and extraction time on the extract efficiency were investigated.The optimal experimental conditions,the polyacrylonitrile fiber was cut into 10 cm pieces,ispropyl alcohol as organic solvent,2.00 ml pH9 NaOH as the donor phase,pH4 HCl as the acceptor phase,1500 r/min of the stirring rate,and 30 min of the extraction time,were selected.An aliquot（20μl）,after extraction,was subsequently withdrawn into a microsyringe and directly injected into an HPLC system with methanol:pH6.0 phosphate buffer（57∶43）as the mobile phase.The C₁₈column was used with flow rate at 0.8 ml/min and temperature at 35℃.The detective wave length was set at 220 nm.Under the optimal conditions high extract efficiency was obtained.Result In Sophora flavescens Ait.and injection preparation:The linear ranges of oxymatrine and matrine were from 11 to 437μg/ml and from 10 to 433μg/ml respectively with the correlation coefficient（r）better than 0.999.The relative standard deviation was lower than 9.4%and 6.7% respectively.The average relative recoveries of oxymatrine and matrine in injection preparation were 83.0%～116.1%and 108.8%～117.8%respectively.The average relative recovery of oxymatrine in Sophora flavescens Ait.was 104.3%～114.7%.The limit of detection was 1.0μg/ml for oxymatrine,1.0μg/ml for matrine.In plasm:The linear ranges of oxymatrine and matrine were from 5 to 119μg/ml and from 3 to 117μg/ml respectively with the correlation coefficient（r）better than 0.997.The relative standard deviation was lower than 5.9%and 5.1%respectively.The average relative recoveries of oxymatrine and matrine were 88.8%～106.4%and 96.0%～104.2%respectively.The limit of detection was 1.4μg/ml for oxymatrine,1.0μg/ml for matrine.In liver:The linear range of matrine was from 3 to 131μg/ml with the correlation coefficient（r）0.9995.The relative standard deviation was lower than 6.8%.The average relative recovery of matrine was 93.7%～101.6%.The limit of detection was 1.0μg/ml for matrine. Conclusion This method applied to eliminate the interference of matrices,had improved the selectivities,and results showed the possibility for the simultaneous determination of oxymatrine and matrine in Sophora flavescens Ait.,injection preparation and biological matrices. Objective Nonaqueous liquid-phase microextraction with back extraction（NLPME/BE）based on porous hollow fibres coupled with high performance liquid chromatography（HPLC）was developed for the simultaneous determination of quercetin,kaempferol and isorhamnetin in Ginkgo Biolbal..Method The effects of kinds of hollow fiber and microextraction solvent,pH in the donor and acceptor phases,stirring rate and extraction time on the extract efficiency were investigated.The optimal experimental conditions,the polysulfone fiber was cut into 10 cm pieces,octanol as organic solvent,pH10 NaOH as acceptor phase,10^-3mol/L HCI of methanol 2.00 ml as the donor phase,1500 r/min of the stirring rate and 30 min of the extraction time,were selected.An aliquot（20μl）,after extraction,was subsequently withdrawn into a mierosyringe and directly injected into an HPLC system with methanol:0.4%H₃PO₄（60∶40）as the mobile phase.The C₁₈ column was used with flow rate at 0.8 ml/min and temperature at 30℃.The detective wave length was set at 360 nm.Under the optimal conditions high extract efficiency was obtained.Result The linear ranges of quercetin,kaempferol and isorhamnetin were from 3.63 to 49.36μg/ml,from 3.63 to 49.36μg/ml and from 1.85 to 25.19μg/ml respectively,with the correlation coefficient（r）better than 0.994.The average relative recoveries were（95.6±4.8）%（n=5）, （107.5±5.3）%（n=5）and（103.4±4.1）%（n=5）respectively.The relative standard deviation was 4.6%,3.7%and 4.3%respectively.The limit of detection was 40,70 and 90ng/ml respectively.Conclusion Nonaqueous liquid-phase microextraction was adopted for the first time.This method was applied to simultaneous determination of quercetin,kaempferol and isorhamnetin in Ginkgo Biolbal..更多还原