HPLC法在国内外药典中的应用与比较

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2/4/20231山东省药品检验所汇报的主要内容一、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2/4/20232山东省药品检验所一、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。2/4/20233山东省药品检验所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2/4/20234山东省药品检验所CHP(2010)：《中国药典》2010年版USP：UnitedStatedPharmacopoeiaEP：EuropeanPharmacopoeiaJP：JapanesePharmacopoeia一、简述2/4/20235山东省药品检验所定义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)<621>：High-pressureliquidchromatography(HPLC),sometimescalledhigh-performanceliquidchromatography,isaseparationtechniquebasedonasolidstationaryphaseandaliquidmobilephase.Separationsareachievedbypartition,adsorption,orion-exchangeprocesses,dependinguponthetypeofstationaryphaseused.2/4/20236山东省药品检验所定义EP(6.0)2.2.29：Liquidchromatographyisamethodofchromatographicseparationbasedonthedifferenceinthedistributionofspeciesbetweentwonon-misciblephases,inwhichpercolatesthroughastationaryphasecontainedinacolumn.2/4/20237山东省药品检验所定义JP(XV)2.01：Liquidchromatographyisamethodtodevelopamixtureinjectedintoacolumnpreparedwithasuitablestationaryphasebypassingaliquidasamobilephasethroughthecolumn,inordertoseparatethemixtureintoitscomponentsbymakinguseofthedifferenceofretentioncapacityagainstthestationaryphase,andtodeterminethecomponents.2/4/20238山东省药品检验所二、高效液相色谱仪基本组成：2/4/20239山东省药品检验所泵CHP(2010)USP(32)EP(6.0)JP(XV)未解释满足等度和梯度洗脱;压力：5000psi或更高;最大可达10ml/min可满足等度或梯度洗脱只强调了恒定速率泵的种类很多，目前应用最多的是柱塞往复泵（恒流泵）2/4/202310山东省药品检验所进样器CHP(2010)USP(32)EP(6.0)JP(XV)未解释微量进样器定量环自动进样器手动

/自动强调重复性好即可2/4/202311山东省药品检验所色谱柱—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3～10μm；<2μm（亚-2μm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：2～8；<2，化学键合相脱落>8，硅胶溶解以5μm最为常见2/4/202312山东省药品检验所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：色谱柱乙烷交联，抵抗高pH侵蚀多点配体键合，抵抗低pH下配体水解pH1~12稳定！KinetexC18柱：通过核-壳颗粒技术，使颗粒减小到2.6μm，而无需高压即可达到提高速度、分离度和灵敏度的效果。2/4/202313山东省药品检验所色谱柱Zorbax

StableBond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pH1.0~8.0，温度上限也可达到80~90℃，甚至在100%水相中也有出色的表现。2/4/202314山东省药品检验所色谱柱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pH2~11.5的范围内是稳定的。2/4/202315山东省药品检验所USP(32)<621>:Stationaryphasesformodern,reverse-phaseliquidchromatographytypicallyconsistofanorganicphasechemicallyboundtosilicaorothermaterials.1.Particles：usually3to10μmindiameter；mayrangeupto50μmormoreforpreparativecolumns.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:2/4/202316山东省药品检验所C-18硅胶柱C-8NH2柱阳离子交换柱（SCX）CN柱苯基柱阴离子交换柱（SAX）2/4/202317山东省药品检验所L57~L67未列出2/4/202318山东省药品检验所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。2/4/202319山东省药品检验所particlesize：3~10μminternaldiameters：prescribedinthemonograph,eg:pH：silicabasedreversed-phasecolumnsareconsideredtobestableinmobilephaseshavinganapparentpHintherange2.0to8.0.temperature:notbeheatedabove60℃Special：EP(6.0)2.2.29：stationaryphasedegradetioncompositionofthemobilephase2/4/202320山东省药品检验所EPMannitolAssay:2/4/202321山东省药品检验所JP(XV)：Acolumnwithastationaryphasechemicallyboundontheinsidewallinsteadofthecolumnpackedwiththepackingmaterialmaybeused.2/4/202322山东省药品检验所检测器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。2/4/202323山东省药品检验所USPandEPUSP(32)EP(6.0)Fixed,variableandmulti-wavelengthdetectorsarewidelyavailable.UV、RID、FLD、ECD......Newdetectorscontinuetobedevelopedinattemptstoovercomethedeficienciesofthosebeingused.UV/Visspectrophotometers,includingdiodearraydetectors,arethemostcommonlyemployeddetectors.2/4/202324山东省药品检验所流动相CHP(2010)反相系统首选甲醇-水系统（采用紫外末端波长检测，首选乙腈-水系统）缓冲盐：少用，尽可能低反相色谱系统，C-18柱，有机相比例应不低于5%。USP(32)High-purityreagentsand“HPLCgrade”organicsolventswater:lowcondutivityandlowUVabsorptionEP(6.0)Fornomal-phasechromatography,lesspolarsolventsareemployed.Wateristobestrictlycontrolled!0.45μmdegassedbysparingwithhelium,sonicationorusingon-linemembrane/vacuumAdjustmentofthepH,iseffectedusingonlytheaqueouscomponentofthemobilephaseandnotthemixture.2/4/202325山东省药品检验所系统适用性SystemSuitability2/4/202326山东省药品检验所定义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.2/4/202327山东省药品检验所DefinitionEP(6.0):Thesystemsuitabilitytestsrepresentanintegralpartofthemethodandareusedtoensureadequateperformanceofthechromatographicsystem.Thevariouscomponentsoftheequipmentemployedmustbequalifiedandbecapableofachievingtheprecisionrequiredtoconductthetestorassay.USP(32):

Systemsuitabilitytestsareanintegralpartofgasandliquidchromatographicmethods.Theyareusedtoverifythattheresolutionandreproducibilityofthechromatographicsystemareadequatefortheanalysistobedone.2/4/202328山东省药品检验所DefinitionUSP(32):

Nosampleanalysisisacceptableunlesstherequirementsofsystemsuitablityhavebeenmet.Sampleanalysesobtainedwhilethesystemfailsrequirementsareunacceptable.Systemsuitabilitymustbedemonstratedthroughouttherunbyinjectionofanappropriatecontrolpreparationatappropriateintervals.Whereverthereisasignificantchangeinequipmentorinacriticalreagent,suitabilitytestingshouldbeperformedbeforetheinjectionofsamples.2/4/202329山东省药品检验所理论板数评价色谱柱的重要指标。CHP(2010)*USP(32)*EP(6.0)JP(XV)n=16(tR/W)2n=5.54(tR/Wh/2)2N=16(t/W)2N=5.54(t/Wh/2)2N=5.54(tR/Wh)2Wh：半高峰宽N=5.54×(tR/W0.5h)2W0.5h：半高峰宽影响因素：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2/4/202330山东省药品检验所分离度（R）衡量色谱系统效能的关键指标！2/4/202331山东省药品检验所分离度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有规定外，应大于1.5

USP(32)*#

EP(6.0)#

JP(XV)*有争议时，以峰宽（W）计算结果为准；#describedinindividualmonograph2/4/202332山东省药品检验所USP(32)Chromatographicsystem—Theliquidchromatographisequippedwitha275-nmdetectoranda4.6-mm×15-cmcolumnthatcontainspackingL1……theresolution,

R,betweentheimpurityCandfamotidinepeaksisnotlessthan1.3;theresolution,R,betweenthefamotidineandimpurityDpeaksisnotlessthan1.3;FamotidineTabletsAssary

2/4/202333山东省药品检验所EP(6.0)左甲状腺素钠含量测定：左羟丙哌嗪对映体纯度：2/4/202334山东省药品检验所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%

。2/4/202335山东省药品检验所重复性

USP(32):Unlessotherwisespecifiedintheindividualmonograph,datafrom

five

replicateinjectionsoftheanalyteareusedtocalculatetherelativestandarddeviation,SR,iftherequirmentis2.0%orless;datafromsix

replicateinjectionsareusediftherelativestandarddeviationrequirmentismorethan2.0%.

2/4/202336山东省药品检验所重复性

EP(6.0):Therepeatabilityofresponseisexpressedasanes

timatedpercentagerelativestandarddeviation(Sr(%))ofaconsecutiveseriesofmeasurementsforNOTfewerthan3injectionsorapplicationsofareferencesolution.meanofindividualvaluesindividualvaluesexpressedaspeakarea,peakheight,orratioofareasbytheinteral

standardisationmethodnumberofindividualvalues2/4/202337山东省药品检验所重复性EP(6.0):Unlessotherwiseprescribed,themaximumpermittedRSDdoesnotexceedtheappropriatevaluegivenintable.Numberofindividualinjections3456B(percent)Maximumpermittedrelativestandarddeviation2.00.410.590.730.852.50.520.740.921.063.00.620.891.101.272/4/202338山东省药品检验所重复性NOTE:Thisrequirementdoesnotapplytotestsforrelatedsubstances.upperlimitgiveninthedefinitionoftheindividualmonographminus100%contant(0.349)numberofreplicateinjectionsofthereferencesolution(3≤n≤6)90%probabilitylevel，n-1degreesoffreedomEP(6.0):Inanassayofanactivesubstancewherethevalueis100percentforapuresubstance,themaximumpermitted(Sr(%)max)fordefinedlimitsiscalculatedusingthefollowingequation:2/4/202339山东省药品检验所重复性

JP(XV):

公式与EP相同。重复次数及限度在各论中要求。例：

RitodrineHydrochlorideRelatedsubsances

Systemsuitability—Testforrequireddetectability:............Systemperformance:............Systemrepeatability:Whenthetestisrepeated6timeswith......，therelativestandarddeviationofthepeakareasofdeferoxamineisnotmorethan3.0%.2/4/202340山东省药品检验所拖尾因子（对称因子）用于评价色谱峰的对称性2/4/202341山东省药品检验所拖尾因子T（orSymmetryfactor）CHP(2010)T=W0.05h/2d1应符合个论项下的规定除另有规定外，峰高法定量时T应在0.95~1.05之间USP(32)T=W0.05/2fEP(6.0)As=W0.05h/2dAnAsvalueof1.0signifiessymmetry.WhenAs＞1.0,thepeakistailing.WhenAs＜1.0,thepeakisfronting.Inarelatedsubstancestestorassay,forapeakinthechromatogramobtainedwithareferencesolutionusedforquantification,thesymmetryfactoris0.8to1.5,unlessotherwiseprescribed.JP(XV)S=W0.05h/2f2/4/202342山东省药品检验所USPItisalsoacommonpracticetomeasuretheAsymmetryfactorastheratioofthedistancebetweentheverticallineconnectingthepeakapexwiththeinterpolatedbaselineandthepeakfront,andthedistancebetweenthatlineandthepeakbackmeasureat10%ofthepeakheight,itwouldbe(W0.10-f0.10)/f0.10However,forthepurposeofUSP,onlytheformulapresentedintheGlossaryofSymbolsisvalid.2/4/202343山东省药品检验所EP中系统适用性：p/vandS/NThepeak-to-valleyratio(p/v)maybeemployedasasystemsuitabilitycriterioninatestforrelatedsubstanceswhenbaselineseparationbetween2peaksisnotachieved.p/v=Hp/HvForexample:Econazolerelatedsubstances2/4/202344山东省药品检验所EPTheshort-termnoiseinfluencestheprecisionofquantification.The

signal-to-noiseratioiscalculatedusingthefollowingequation:S/N=2H/h

Forexample:

ketotifenhydrogenfumarate

relatedsubstances2/4/202345山东省药品检验所色谱条件的调整AdjustmentofChromatographicConditions2/4/202346山东省药品检验所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。2/4/202347山东省药品检验所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。2/4/202348山东省药品检验所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：82.5:17.5~67.5:32.5解决：70：302/4/202349山东省药品检验所USP(32)pHofmobilephaseconcentrationofsaltsinbufferratioofcomponentsinmobilephaseWavelengthofUVdetectorinjectionvolumepHoftheaqueousbufferwithin±0.2unitsofthevalueorrangespecifiedwithin±10%providedthepermittedpHvariationismetapplytominorcomponentscanbeadjustedby±30％relativecannotexceed±10%absolute,seeexample:binaryorternarymixturesnotpermittedcanbereducedasfarasisconsistentwithacceptedprecisionanddetectionlimitscolumnlengthcolumninnerdiameterparticlesizeflowratecolumntemperature±70%±25%canbereducedbyasmuchas50%±50%±10°

Ifadjustmentsofoperatingconditionstomeetsystemsuitabilityrequirments

arenecessary,eachofthefollowingisthemaxiumvariationthatcanbeconsidered,unlessotherwisedirectedinthemonograph.2/4/202350山东省药品检验所USP(32)BinaryMixtures—SPECIFIEDRATIOOF50:50—Thirtypercentof50is15%absolute,butthisexceedsthemaximumpermittedchangeof±10%absoluteineithercomponent.Therefore,themobilephaseratiomaybeadjustedonlywithintherangeof40:60to60:40.

SPECIFIEDRATIOOF2:98—Thirtypercentof2is0.6%absolute.Thereforethemaximumallowedadjustmentiswithintherangeof1.4:98.6to2.6:97.4.

2/4/202351山东省药品检验所USP(32)TernaryMixtures—SPECIFIEDRATIOOF60:35:5—Forthesecondcomponent,30%of35is10.5%absolute,whichexceedsthemaximumpermittedchangeof±10%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.2/4/202352山东省药品检验所EP(6.0)Theextenttowhichthevariousparametersofachromatographictestmaybeadjustedtosatisfythesystemsuitabilitycriteriawithoutfundamentallymodifyingthemethodsarelistedbelow.Isocraticelution:pHoftheaqueouscomponentofthemobilephaseconcentrationofsaltsinbuffercompositionofthemobilephaseWavelengthcolumntemperature±0.2pH±1.0pH(non-ionisable)

within±10%Minorcomponent30%relativeor±2%absolutewhicheverislarger.Nocomponentcanbealteredbymorethan10%absolute（seeexample）noadjustmentpermitted±10℃columnlengthinteraldiameterparticlesizeflowrateinjectionvolume±70%±25%maximumreductionof50%noincreasepermitted±50%alargeradjustmentisacceptable（seeexample）maybedecreasednoincreasepermitted2/4/202353山东省药品检验所EP(6.0)Compositionofthemobilephase:theamountoftheminorsolventcomponentmaybeadjustedby±30%relativeor±2%absolute,whicheveristhelarger;foraminorcomponentat10%ofthemobilephase,a30%relativeadjustmentallowsarangeof7-13%

wherea

2%absoluteadjustmentallowsarangeof8-12%,therelativevaluebeingthereforethelarger;Foraminorcomponentat5%ofthemobilephase,a30%relativeadjustmentallowsarangeof3.5-6.5%whereasa2%absoluteadjustment

allowsarangeof

3-7%,theabsolutevaluebeinginthiscasethelarger.Noothercomponentisalteredbymorethan10%absolute.2/4/202354山东省药品检验所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2/4/202355山东省药品检验所EP(6.0)pHoftheaqueouscomponentofthemobilephaseconcentrationofsaltsinbuffercompositionofthemobilephaseWavelengthofUVdetectorcolumntemperaturenoadjustmentpermitted

noadjustmentpermittedthesystemsuitabilityrequirementsarefulfilleldtheprincipalpeakelutewithin±15%oftheindicatedretentiontimeelutionpowerisnotweakerthanbeforenoadjustmentpermitted±5℃columnlengthinteraldiameterparticlesizeflowrateinjectionvolume±70%±25%noadjustmentpermittedsameasisocraticelutionmaybedecreasednoincreasepermittedGradientelution：morecriticalthanwithisocraticelution2/4/202356山东省药品检验所HPLC的应用及方法开发2/4/202357山东省药品检验所HPLC应用类型定

义I原料药或药物制剂活性成分中主要成分的分析程序-如含量测定II原料药中杂质检查或药物制剂中降解产物的分析程序，这些分析程序包括定量试验或限度试验III用于性能参数测试的分析程序-如溶出度、释放度IV鉴别试验HPLC可以用来干什么？2/4/202358山东省药品检验所方法开发的指导原则ICHUSP《中国药典》2/4/202359山东省药品检验所ICHICH(人用药品注册技术要求国际协调会)三方协调指导原则

Q2A：分析方法论证的文本ICH三方协调指导原则

Q2B：方法学ICH三方协调指导原则2/4/202360山东省药品检验所ICH分析方法的类型项目鉴别杂质检查含量分析，溶出度，含量/效价定量限度检测准确