I武汉大学药学院本科生毕业论文磷脂的纯化及毛细管电泳定性定量分析学生姓名:何秀峰学号:200132290015指导老师姓名、职称:肖玉秀、副教授2005年05月IIPurificationofPhospholipidsandDeterminationofPhosphatidylcholinebyHighPerformanceCapillaryElectrophoresisHeXiufengDirectedbyAssociateProfessorXiaoYuxiuCollegeofPharmacyWuhanUniversityIIIMay,2005摘要目的:通过溶剂萃取和柱色谱法,从大豆粉末磷脂中分离纯化卵磷脂(PC),为脂质体制备提供高纯载体材料;建立磷脂的高效毛细管电泳(HPCE)定性定量分析方法。方法:①将溶剂萃取法与柱色谱法相结合从大豆粉末磷脂中分离纯化PC。先用95%乙醇浸提粉末磷脂得到粗磷脂,然后以硅胶为吸附介质,甲醇为洗脱剂等度淋洗,对粗磷脂继续进行柱色谱分离纯化,得到较高纯度的PC。采用高效快速的薄层色谱法(TLC)对分离纯化过程进行监测,以2.5cm×7.5cm硅胶G为薄层板,最优化展开剂为氯仿-甲醇-水(65:25:5,V/V)。参照磷脂通用技术条件及中国药典,对PC产品的红外光谱特征、氧化程度以及其它相关质量指标进行考察。②采用HPCE法对磷脂进行分离分析,并与高效液相色谱(HPLC)法比较。以50μmI.D.×375μmO.D.(总长60cm,有效长度50cm)熔融石英毛细管为分离通道,35mmol·L1脱氧胆酸钠(SDC)1mmol•L1硼砂43%(V/V)正丙醇(pH8.30)为电泳缓冲液,操作电压25kV,温度44℃,紫外检测波长200nm,外标法定量。结果:①TLC法能实现粗磷脂中各组分的良好分离。从大豆粉末磷脂中分离纯化得到的PC,其红外光谱特征与标准品一致,含量高达92.8%,氧化指数0.056,水分及挥发物1.28%,乙醇可溶物99.84%,酸价8.35mgKOH/g,碘价92.3gI/100g,350nm处的吸收度为0.12。②HPCE法能完全分离5种磷脂组分,定量分析PC的标准曲线为A=-1.40376+0.34166×C(r=0.9990),线性范围0.1mg•mL-1~1mg•mL-1,检测限2.56μg•mL1,定量限8.51μg•mL1,日内、日间定量精密度分别为0.695%~2.12%;1.39%~5.47%,回收率为96.7%~99.7%。结论:溶剂萃取法与柱色谱法相结合,可从大豆粉末磷脂中分离出高纯度PC,TLC法能很好地监测分离纯化过程;所制PC的红外光谱特征与标准品一致,含量高,氧化程度低,其它各项质量指标均符合磷脂通用技术条件中一级产品的规定,可作为制备脂质体的高纯载体材料。所建立的HPCE法对磷脂各成分的分离效果良好;用于定量分析PC,准确度高、重现性好,可作为PC的质量控制手段。IV关键词:磷脂;柱色谱纯化;TLC;毛细管电泳AbstractObjective:Purificationofphosphatidylcholine(PC)fromsoybeanphospholipidsbyusingcolumnchromatographytopreparetheliposomescarriermaterial.Andestablishmentofananalyticalmethodofhighperformancecapillaryelectrophoresis(HPCE)fordeterminationofPC.Methods:①Preparationthehighpurelecithinfromsoybeanphospholipidsthroughsolventsextractionandcolumnchromatography.Thecrudephospholipidsareattainedbyusing95%ethanolextractionfromsoybeanphospholipids.Thecrudephospholipidsareusedastherawmaterialsforchromatographyseparationonasilicacolumn,highpurePCisobtainedwhenthecrudephospholipidsareisocraticlyelutedbymethanol.AhighefficientandfastTLCmethodforqualitativeanalysisisusedforcolumnchromatogramon2.5cm×7.5cmsilicaGthinlayerplatewhileusingchloroformmethanolwater(65:25:5,V/V)astheoptimizeddeveloper.Accordingtopharmacopoeiaandindustrystandard,IRspectrum,oxidationindexandtheotherqualityofthepurifiedPCarestudied.②ThemethodofquantitativeanalysisofPCbyHPCEisestablishedandcomparedtoHPLC.Thedeterminationisperformedona50μmI.D.×375μmO.D.fusedsilicacapillary(60cmintotallengthand50cmtothedetector).Optimizedanalyticalconditionsareasfollows:runningbuffersystemof35mmol•L1SDC1mmol•L1boraxbuffer43%(V/V)npropanolwithpH8.30,25kVappliedvoltage,temperature44℃andUVdetectionat200nm.Calibrat...