HPLC法在国内外药典中的应用与比较.ppt

HPLC法在国内外药典中的应用与比较,山东省药品检验所化学药品科王小兵,2019/12/19,山东省药品检验所,2,汇报的主要内容,一、简述二、高效液相色谱仪三、系统适用性四、色谱条件的调整五、HPLC的应用及方法开发,2019/12/19,山东省药品检验所,3,一、简述,中国药典2010年版二部共收载2271个品种，新增品种330个，修订品种1500个，涉及HPLC检测项目的品种有1291个，占总品种的57%，其中新增/修订926个。HPLC法在整个药典品种的检验中占有重要地位。,2019/12/19,山东省药品检验所,4,HighPerformanceLiquidChromatography简称HPLC，开始应用于20世纪60年代后期，现已趋于成熟，广泛应用于医药、生化、天然产物主要组分分析，以及食品、化妆品分析，环境分析，农业分析，石油化工分析等。优点：高压+高速+高效+高灵敏度,一、简述,2019/12/19,山东省药品检验所,5,CHP(2010)：中国药典2010年版USP：UnitedStatedPharmacopoeiaEP：EuropeanPharmacopoeiaJP：JapanesePharmacopoeia,一、简述,2019/12/19,山东省药品检验所,6,定义,CHP(2010)附录VD：HPLC系采用高压输液泵将规定的流动相泵入装有填充剂的色谱柱，对供试品进行分离测定的色谱方法。USP(32)：High-pressureliquidchromatography(HPLC),sometimescalledhigh-performanceliquidchromatography,isaseparationtechniquebasedonasolidstationaryphaseandaliquidmobilephase.Separationsareachievedbypartition,adsorption,orion-exchangeprocesses,dependinguponthetypeofstationaryphaseused.,2019/12/19,山东省药品检验所,7,定义,EP(6.0)2.2.29：Liquidchromatographyisamethodofchromatographicseparationbasedonthedifferenceinthedistributionofspeciesbetweentwonon-misciblephases,inwhichpercolatesthroughastationaryphasecontainedinacolumn.,2019/12/19,山东省药品检验所,8,定义,JP(XV)2.01：Liquidchromatographyisamethodtodevelopamixtureinjectedintoacolumnpreparedwithasuitablestationaryphasebypassingaliquidasamobilephasethroughthecolumn,inordertoseparatethemixtureintoitscomponentsbymakinguseofthedifferenceofretentioncapacityagainstthestationaryphase,andtodeterminethecomponents.,2019/12/19,山东省药品检验所,9,二、高效液相色谱仪,基本组成：,2019/12/19,山东省药品检验所,10,泵,泵的种类很多，目前应用最多的是柱塞往复泵（恒流泵）,2019/12/19,山东省药品检验所,11,进样器,2019/12/19,山东省药品检验所,12,色谱柱分离的核心,CHP(2010)附录：1.正相：硅胶柱；反相：C-18柱、C-8柱；2.粒径：普通310m；2m（亚-2m仅能用于UPLC）；3.温度要求：以硅胶为载体的通常40；不宜超过60；4.pH要求：28；,8，硅胶溶解,以5m最为常见,2019/12/19,山东省药品检验所,13,色谱柱技术发展很快，新填料新技术也不断涌现，给我们提供了更多的选择。Gemini-NXC18柱：,色谱柱,KinetexC18柱：通过核-壳颗粒技术，使颗粒减小到2.6m，而无需高压即可达到提高速度、分离度和灵敏度的效果。,2019/12/19,山东省药品检验所,14,色谱柱,ZorbaxStableBond柱：采用了较大的二异丁基(SB-C18)或二异丙基(SB-C8、SB-C3、SB-Phenyl、SB-CN、SB-Aq)侧链基团和空间位阻，避免了在低pH条件下的水解破坏，其pH1.08.0，温度上限也可达到8090，甚至在100%水相中也有出色的表现。,2019/12/19,山东省药品检验所,15,色谱柱,ZorbaxExtendC18柱：采用独特的双配位C18-C18键合技术，使的在高pH条件下使用硅胶基色谱柱成为可能，在pH211.5的范围内是稳定的。,2019/12/19,山东省药品检验所,16,USP(32):,Stationaryphasesformodern,reverse-phaseliquidchromatographytypicallyconsistofanorganicphasechemicallyboundtosilicaorothermaterials.1.Particles：usually3to10mindiameter；mayrangeupto50mormoreforpreparativecolumns.2.Internaldiameters：usually2to5mmforanalyticalseparation，lagerforpreparativechromatography.3.Temperature:onlyrarelyabove60；4.根据填料不同，USP在ChromatographicreagentsintheReagents,Indicators,andSolutionssection列出了USP-NF所用到的不同类型的柱子，如下所示:,C-18,硅胶柱,C-8,NH2柱,阳离子交换柱（SCX）,CN柱,苯基柱,阴离子交换柱（SAX）,L57L67未列出,2019/12/19,山东省药品检验所,19,EP(6.0)2.2.29：,大部分分离机制都是基于以化学键合硅胶作为固定相，极性溶剂作为流动相的色谱条件。而化学键合相的性质往往决定了色谱系统的分离性能。,2019/12/19,山东省药品检验所,20,particlesize：310minternaldiameters：prescribedinthemonograph,eg:pH：silicabasedreversed-phasecolumnsareconsideredtobestableinmobilephaseshavinganapparentpHintherange2.0to8.0.temperature:notbeheatedabove60Special：,EP(6.0)2.2.29：,2019/12/19,山东省药品检验所,21,EP,MannitolAssay:,2019/12/19,山东省药品检验所,22,JP(XV)：,Acolumnwithastationaryphasechemicallyboundontheinsidewallinsteadofthecolumnpackedwiththepackingmaterialmaybeused.,2019/12/19,山东省药品检验所,23,检测器,CHP(2010):UVDADFLDECDELSDRIDMS,优点：灵敏度高、噪音低、线性范围宽、对流速和温度的波动不灵敏，适用于梯度洗脱及制备色谱。缺点：只能检测有紫外吸收的物质，流动相的选择有一定限制，流动相的截止波长必须小于检测波长。适用范围：大多数有紫外吸收的化合物。,优点：通用型检测器，对各种物质有几乎相同的响应。缺点：灵敏度相对较低，流动相必须是挥发性的，不能用非挥发性的缓冲盐及表面活性剂。适用范围：适用于挥发性低于流动相的组分，主要用于糖类、高级脂肪酸、磷脂、维生素、氨基酸、甘油三酯、皂苷及甾体等等无紫外吸收或紫外末端吸收的化合物的检测。,优点：灵敏度高、选择性好，是微量组分和体内药物分析常用的检测器之一。缺点：只适用于能够产生荧光的物质的检测，适用范围不如紫外检测器。影响因素较多。适用范围：具有天然荧光的物质或通过荧光衍生化变成荧光衍生物后测定，主要用于氨基酸、多环芳烃、维生素、甾体化合物及酶等的检测。,优点：灵敏度很高，尤其适用于痕量组分分析。缺点：干扰比较多，对温度和流速的变化比较敏感。适用范围：应用范围广，凡具氧化还原活性的或经衍生化后具氧化还原活性的物质。,优点：灵敏度高，选择性好，能同时给出组分的结构信息。缺点：响应信号受离子化效率限制，仪器较为昂贵，流动相必须是挥发性的，不能用非挥发性的缓冲盐及表面活性剂。适用范围：组分的结构鉴别，微量及痕量组分的分析，药物代谢分析等。,优点：通用型检测器，只要组分的折光率与流动相的折光率有足够的差别就能检测。缺点：灵敏度低、受环境温度、流量及流动相组成等波动的影响大，一般不能用于梯度洗脱。适用范围：RID为通用型检测器，适用于无紫外吸收化合物的分析，如糖类分析。,2019/12/19,山东省药品检验所,24,USPandEP,2019/12/19,山东省药品检验所,25,流动相,2019/12/19,山东省药品检验所,26,系统适用性SystemSuitability,2019/12/19,山东省药品检验所,27,定义,CHP(2010):色谱系统的适用性试验通常包括理论板数、分离度、重复性和拖尾因子四个参数，其中，分离度和重复性尤为重要。ICHdefinition:Systemsuitabilitytestingisanintegralpartofmanyanalyticalprocedures.Thetestsarebasedontheconceptthattheequipment,electronics,analyticaloperationsandsamplestobeanalyzedconstituteanintegralsystemthatcanbeevaluatedassuch.,2019/12/19,山东省药品检验所,28,Definition,EP(6.0):Thesystemsuitabilitytestsrepresentanintegralpartofthemethodandareusedtoensureadequateperformanceofthechromatographicsystem.Thevariouscomponentsoftheequipmentemployedmustbequalifiedandbecapableofachievingtheprecisionrequiredtoconductthetestorassay.USP(32):Systemsuitabilitytestsareanintegralpartofgasandliquidchromatographicmethods.Theyareusedtoverifythattheresolutionandreproducibilityofthechromatographicsystemareadequatefortheanalysistobedone.,2019/12/19,山东省药品检验所,29,Definition,USP(32):Nosampleanalysisisacceptableunlesstherequirementsofsystemsuitablityhavebeenmet.Sampleanalysesobtainedwhilethesystemfailsrequirementsareunacceptable.Systemsuitabilitymustbedemonstratedthroughouttherunbyinjectionofanappropriatecontrolpreparationatappropriateintervals.Whereverthereisasignificantchangeinequipmentorinacriticalreagent,suitabilitytestingshouldbeperformedbeforetheinjectionofsamples.,2019/12/19,山东省药品检验所,30,理论板数,评价色谱柱的重要指标。,影响因素：固定相、柱温、流动相和保留时间。*有争议时，以峰宽（W）计算结果为准,2019/12/19,山东省药品检验所,31,分离度（R）,衡量色谱系统效能的关键指标！,2019/12/19,山东省药品检验所,32,分离度（R）,*有争议时，以峰宽（W）计算结果为准；#describedinindividualmonograph,2019/12/19,山东省药品检验所,33,USP(32),ChromatographicsystemTheliquidchromatographisequippedwitha275-nmdetectoranda4.6-mm15-cmcolumnthatcontainspackingL1theresolution,R,betweentheimpurityCandfamotidinepeaksisnotlessthan1.3;theresolution,R,betweenthefamotidineandimpurityDpeaksisnotlessthan1.3;,FamotidineTabletsAssary,2019/12/19,山东省药品检验所,34,EP(6.0),左甲状腺素钠含量测定：,左羟丙哌嗪对映体纯度：,2019/12/19,山东省药品检验所,35,重复性,评价连续进样中，色谱系统响应值的重复性能。CHP(2010):外标法：对照品溶液，连续进样5次，峰面积RSD不得过2.0%。内标法：配制相当于80%、100%、120%的对照品溶液，加入内标溶液，分别至少进样2次，计算平均校正因子的RSD不得过2.0%。,2019/12/19,山东省药品检验所,36,重复性,USP(32):Unlessotherwisespecifiedintheindividualmonograph,datafromfivereplicateinjectionsoftheanalyteareusedtocalculatetherelativestandarddeviation,SR,iftherequirmentis2.0%orless;datafromsixreplicateinjectionsareusediftherelativestandarddeviationrequirmentismorethan2.0%.,2019/12/19,山东省药品检验所,37,重复性,EP(6.0):Therepeatabilityofresponseisexpressedasanestimatedpercentagerelativestandarddeviation(Sr(%)ofaconsecutiveseriesofmeasurementsforNOTfewerthan3injectionsorapplicationsofareferencesolution.,meanofindividualvalues,individualvaluesexpressedaspeakarea,peakheight,orratioofareasbytheinteralstandardisationmethod,numberofindividualvalues,2019/12/19,山东省药品检验所,38,重复性,EP(6.0):Unlessotherwiseprescribed,themaximumpermittedRSDdoesnotexceedtheappropriatevaluegivenintable.,2019/12/19,山东省药品检验所,39,重复性,NOTE:Thisrequirementdoesnotapplytotestsforrelatedsubstances.,upperlimitgiveninthedefinitionoftheindividualmonographminus100%,contant(0.349),numberofreplicateinjectionsofthereferencesolution(3n6),90%probabilitylevel，n-1degreesoffreedom,EP(6.0):Inanassayofanactivesubstancewherethevalueis100percentforapuresubstance,themaximumpermitted(Sr(%)max)fordefinedlimitsiscalculatedusingthefollowingequation:,2019/12/19,山东省药品检验所,40,重复性,JP(XV):公式与EP相同。重复次数及限度在各论中要求。例：RitodrineHydrochlorideRelatedsubsancesSystemsuitabilityTestforrequireddetectability:.Systemperformance:.Systemrepeatability:Whenthetestisrepeated6timeswith.，therelativestandarddeviationofthepeakareasofdeferoxamineisnotmorethan3.0%.,2019/12/19,山东省药品检验所,41,拖尾因子（对称因子）,用于评价色谱峰的对称性,2019/12/19,山东省药品检验所,42,拖尾因子T（orSymmetryfactor）,2019/12/19,山东省药品检验所,43,USP,ItisalsoacommonpracticetomeasuretheAsymmetryfactorastheratioofthedistancebetweentheverticallineconnectingthepeakapexwiththeinterpolatedbaselineandthepeakfront,andthedistancebetweenthatlineandthepeakbackmeasureat10%ofthepeakheight,itwouldbe(W0.10-f0.10)/f0.10However,forthepurposeofUSP,onlytheformulapresentedintheGlossaryofSymbolsisvalid.,2019/12/19,山东省药品检验所,44,EP中系统适用性：p/vandS/N,Thepeak-to-valleyratio(p/v)maybeemployedasasystemsuitabilitycriterioninatestforrelatedsubstanceswhenbaselineseparationbetween2peaksisnotachieved.p/v=Hp/Hv,Forexample:Econazolerelatedsubstances,2019/12/19,山东省药品检验所,45,EP,Theshort-termnoiseinfluencestheprecisionofquantification.Thesignal-to-noiseratioiscalculatedusingthefollowingequation:S/N=2H/h,Forexample:ketotifenhydrogenfumaraterelatedsubstances,2019/12/19,山东省药品检验所,46,色谱条件的调整AdjustmentofChromatographicConditions,2019/12/19,山东省药品检验所,47,CHP(2010),明确规定：不可变的有：固定相的种类、流动相的组分、检测器类型可变的有：色谱柱内径、长度、载体粒度、流动相流速、混合流动相各组分的比例、柱温、进样量、检测器的灵敏度。,2019/12/19,山东省药品检验所,48,CHP(2010),与CHP(2005)不同：中国药典2010年版规定了流动相调整的限度。调整流动相组分比例时，以组分比例较低者（50）相对于自身改变量不超过30且相对于总量的改变量不超过10为限，如30相对改变量的数值超过总量的10时，则改变量以总量的10为限。,2019/12/19,山东省药品检验所,49,CHP(2010),例：奥美拉唑肠溶片，释放度检查，色谱条件：,问题：峰型差！流动相可调范围：82.5:17.567.5:32.5解决：70：30,2019/12/19,山东省药品检验所,50,USP(32),Ifadjustmentsofoperatingconditionstomeetsystemsuitabilityrequirmentsarenecessary,eachofthefollowingisthemaxiumvariationthatcanbeconsidered,unlessotherwisedirectedinthemonograph.,2019/12/19,山东省药品检验所,51,USP(32),BinaryMixturesSPECIFIEDRATIOOF50:50Thirtypercentof50is15%absolute,butthisexceedsthemaximumpermittedchangeof10%absoluteineithercomponent.Therefore,themobilephaseratiomaybeadjustedonlywithintherangeof40:60to60:40.SPECIFIEDRATIOOF2:98Thirtypercentof2is0.6%absolute.Thereforethemaximumallowedadjustmentiswithintherangeof1.4:98.6to2.6:97.4.,2019/12/19,山东省药品检验所,52,USP(32),TernaryMixturesSPECIFIEDRATIOOF60:35:5Forthesecondcomponent,30%of35is10.5%absolute,whichexceedsthemaximumpermittedchangeof10%absoluteinanycomponent.Thereforethesecondcomponentmaybeadjustedonlywithintherangeof25%to45%absolute.Forthethirdcomponent,30%of5is1.5%absolute.Inallcases,asufficientquantityofthefirstcomponentisusedtogiveatotalof100%.Therefore,mixturerangesof50:45:5to70:25:5or58.5:35:6.5to61.5:35:3.5wouldmeettherequirement.,2019/12/19,山东省药品检验所,53,EP(6.0),Theextenttowhichthevariousparametersofachromatographictestmaybeadjustedtosatisfythesystemsuitabilitycriteriawithoutfundamentallymodifyingthemethodsarelistedbelow.Isocraticelution:,2019/12/19,山东省药品检验所,54,EP(6.0),Compositionofthemobilephase:theamountoftheminorsolventcomponentmaybeadjustedby30%relativeor2%absolute,whicheveristhelarger;foraminorcomponentat10%ofthemobilephase,a30%relativeadjustmentallowsarangeof7-13%wherea2%absoluteadjustmentallowsarangeof8-12%,therelativevaluebeingthereforethelarger;Foraminorcomponentat5%ofthemobilephase,a30%relativeadjustmentallowsarangeof3.5-6.5%whereasa2%absoluteadjustmentallowsarangeof3-7%,theabsolutevaluebeinginthiscasethelarger.Noothercomponentisalteredbymorethan10%absolute.,2019/12/19,山东省药品检验所,55,EP(6.0),Whencolumndimensionsarechanged,theflowratemaybeajustedasnecessaryusingequation:,F1：规定的流速F2：调整后的流速l1：规定的柱长l2：实际所用的柱长d1：规定柱子的内径d2：实际所用柱子的内径,2019/12/19,山东省药品检验所,56,EP(6.0),Gradientelution：morecriticalthanwithisocraticelution,2019/12/19,山东省药品检验所,57,HPLC的应用及方法开发,2019/12/19,山东省药品检验所,58,HPLC应用,HPLC可以用来干什么？,2019/12/19,山东省药品检验