HPLC法在國內(nèi)外藥典中的應用與比較

HPLC法在國內(nèi)外藥典中的

應用與比較

山東省藥品檢驗所化學藥品科王小兵2/4/20231山東省藥品檢驗所匯報的主要內(nèi)容一、簡述二、高效液相色譜儀三、系統(tǒng)適用性四、色譜條件的調(diào)整五、HPLC的應用及方法開發(fā)2/4/20232山東省藥品檢驗所一、簡述《中國藥典》2010年版二部共收載2271個品種，新增品種330個，修訂品種1500個，涉及HPLC檢測項目的品種有1291個，占總品種的57%，其中新增/修訂926個。HPLC法在整個藥典品種的檢驗中占有重要地位。2/4/20233山東省藥品檢驗所HighPerformanceLiquidChromatography簡稱HPLC，開始應用于20世紀60年代后期，現(xiàn)已趨于成熟，廣泛應用于醫(yī)藥、生化、天然產(chǎn)物主要組分分析，以及食品、化妝品分析，環(huán)境分析，農(nóng)業(yè)分析，石油化工分析等。優(yōu)點：高壓+高速+高效+高靈敏度一、簡述2/4/20234山東省藥品檢驗所CHP(2010)：《中國藥典》2010年版USP：UnitedStatedPharmacopoeiaEP：EuropeanPharmacopoeiaJP：JapanesePharmacopoeia一、簡述2/4/20235山東省藥品檢驗所定義CHP(2010)附錄VD：HPLC系采用高壓輸液泵將規(guī)定的流動相泵入裝有填充劑的色譜柱，對供試品進行分離測定的色譜方法。USP(32)<621>：High-pressureliquidchromatography(HPLC),sometimescalledhigh-performanceliquidchromatography,isaseparationtechniquebasedonasolidstationaryphaseandaliquidmobilephase.Separationsareachievedbypartition,adsorption,orion-exchangeprocesses,dependinguponthetypeofstationaryphaseused.2/4/20236山東省藥品檢驗所定義EP(6.0)2.2.29：Liquidchromatographyisamethodofchromatographicseparationbasedonthedifferenceinthedistributionofspeciesbetweentwonon-misciblephases,inwhichpercolatesthroughastationaryphasecontainedinacolumn.2/4/20237山東省藥品檢驗所定義JP(XV)2.01：Liquidchromatographyisamethodtodevelopamixtureinjectedintoacolumnpreparedwithasuitablestationaryphasebypassingaliquidasamobilephasethroughthecolumn,inordertoseparatethemixtureintoitscomponentsbymakinguseofthedifferenceofretentioncapacityagainstthestationaryphase,andtodeterminethecomponents.2/4/20238山東省藥品檢驗所二、高效液相色譜儀基本組成：2/4/20239山東省藥品檢驗所泵CHP(2010)USP(32)EP(6.0)JP(XV)未解釋滿足等度和梯度洗脫;壓力：5000psi或更高;最大可達10ml/min可滿足等度或梯度洗脫只強調(diào)了恒定速率泵的種類很多，目前應用最多的是柱塞往復泵（恒流泵）2/4/202310山東省藥品檢驗所進樣器CHP(2010)USP(32)EP(6.0)JP(XV)未解釋微量進樣器定量環(huán)自動進樣器手動

/自動強調(diào)重復性好即可2/4/202311山東省藥品檢驗所色譜柱—分離的核心CHP(2010)附錄：1.正相：硅膠柱；反相：C-18柱、C-8柱；2.粒徑：普通3～10μm；<2μm（亞-2μm僅能用于UPLC）；3.溫度要求：以硅膠為載體的通常<40℃；不宜超過60℃；4.pH要求：2～8；<2，化學鍵合相脫落>8，硅膠溶解以5μm最為常見2/4/202312山東省藥品檢驗所色譜柱技術發(fā)展很快，新填料新技術也不斷涌現(xiàn)，給我們提供了更多的選擇。Gemini-NXC18柱：色譜柱乙烷交聯(lián)，抵抗高pH侵蝕多點配體鍵合，抵抗低pH下配體水解pH1~12穩(wěn)定！KinetexC18柱：通過核-殼顆粒技術，使顆粒減小到2.6μm，而無需高壓即可達到提高速度、分離度和靈敏度的效果。2/4/202313山東省藥品檢驗所色譜柱Zorbax

StableBond柱：采用了較大的二異丁基(SB-C18)或二異丙基(SB-C8、SB-C3、SB-Phenyl、SB-CN、SB-Aq)側(cè)鏈基團和空間位阻，避免了在低pH條件下的水解破壞，其pH1.0~8.0，溫度上限也可達到80~90℃，甚至在100%水相中也有出色的表現(xiàn)。2/4/202314山東省藥品檢驗所色譜柱ZorbaxExtendC18柱：采用獨特的雙配位C18-C18鍵合技術，使的在高pH條件下使用硅膠基色譜柱成為可能，在pH2~11.5的范圍內(nèi)是穩(wěn)定的。2/4/202315山東省藥品檢驗所USP(32)<621>:Stationaryphasesformodern,reverse-phaseliquidchromatographytypicallyconsistofanorganicphasechemicallyboundtosilicaorothermaterials.1.Particles：usually3to10μmindiameter；mayrangeupto50μmormoreforpreparativecolumns.2.Internaldiameters：usually2to5mmforanalyticalseparation，lagerforpreparativechromatography.3.Temperature:onlyrarelyabove60℃；4.根據(jù)填料不同，USP在ChromatographicreagentsintheReagents,Indicators,andSolutionssection列出了USP-NF所用到的不同類型的柱子，如下所示:2/4/202316山東省藥品檢驗所C-18硅膠柱C-8NH2柱陽離子交換柱（SCX）CN柱苯基柱陰離子交換柱（SAX）2/4/202317山東省藥品檢驗所L57~L67未列出2/4/202318山東省藥品檢驗所EP(6.0)2.2.29：大部分分離機制都是基于以化學鍵合硅膠作為固定相，極性溶劑作為流動相的色譜條件。而化學鍵合相的性質(zhì)往往決定了色譜系統(tǒng)的分離性能。2/4/202319山東省藥品檢驗所particlesize：3~10μminternaldiameters：prescribedinthemonograph,eg:pH：silicabasedreversed-phasecolumnsareconsideredtobestableinmobilephaseshavinganapparentpHintherange2.0to8.0.temperature:notbeheatedabove60℃Special：EP(6.0)2.2.29：stationaryphasedegradetioncompositionofthemobilephase2/4/202320山東省藥品檢驗所EPMannitolAssay:2/4/202321山東省藥品檢驗所JP(XV)：Acolumnwithastationaryphasechemicallyboundontheinsidewallinsteadofthecolumnpackedwiththepackingmaterialmaybeused.2/4/202322山東省藥品檢驗所檢測器CHP(2010):UVDADFLDECDELSDRIDMS優(yōu)點：靈敏度高、噪音低、線性范圍寬、對流速和溫度的波動不靈敏，適用于梯度洗脫及制備色譜。

缺點：只能檢測有紫外吸收的物質(zhì)，流動相的選擇有一定限制，流動相的截止波長必須小于檢測波長。適用范圍：大多數(shù)有紫外吸收的化合物。優(yōu)點：通用型檢測器，對各種物質(zhì)有幾乎相同的響應。缺點：靈敏度相對較低，流動相必須是揮發(fā)性的，不能用非揮發(fā)性的緩沖鹽及表面活性劑。適用范圍：適用于揮發(fā)性低于流動相的組分，主要用于糖類、高級脂肪酸、磷脂、維生素、氨基酸、甘油三酯、皂苷及甾體等等無紫外吸收或紫外末端吸收的化合物的檢測。優(yōu)點：靈敏度高、選擇性好，是微量組分和體內(nèi)藥物分析常用的檢測器之一。缺點：只適用于能夠產(chǎn)生熒光的物質(zhì)的檢測，適用范圍不如紫外檢測器。影響因素較多。適用范圍：具有天然熒光的物質(zhì)或通過熒光衍生化變成熒光衍生物后測定，主要用于氨基酸、多環(huán)芳烴、維生素、甾體化合物及酶等的檢測。優(yōu)點：靈敏度很高，尤其適用于痕量組分分析。缺點：干擾比較多，對溫度和流速的變化比較敏感。適用范圍：應用范圍廣，凡具氧化還原活性的或經(jīng)衍生化后具氧化還原活性的物質(zhì)。優(yōu)點：靈敏度高，選擇性好，能同時給出組分的結(jié)構(gòu)信息。缺點：響應信號受離子化效率限制，儀器較為昂貴，流動相必須是揮發(fā)性的，不能用非揮發(fā)性的緩沖鹽及表面活性劑。適用范圍：組分的結(jié)構(gòu)鑒別，微量及痕量組分的分析，藥物代謝分析等。優(yōu)點：通用型檢測器，只要組分的折光率與流動相的折光率有足夠的差別就能檢測。缺點：靈敏度低、受環(huán)境溫度、流量及流動相組成等波動的影響大，一般不能用于梯度洗脫。適用范圍：RID為通用型檢測器，適用于無紫外吸收化合物的分析，如糖類分析。2/4/202323山東省藥品檢驗所USPandEPUSP(32)EP(6.0)Fixed,variableandmulti-wavelengthdetectorsarewidelyavailable.UV、RID、FLD、ECD......Newdetectorscontinuetobedevelopedinattemptstoovercomethedeficienciesofthosebeingused.UV/Visspectrophotometers,includingdiodearraydetectors,arethemostcommonlyemployeddetectors.2/4/202324山東省藥品檢驗所流動相CHP(2010)反相系統(tǒng)首選甲醇-水系統(tǒng)（采用紫外末端波長檢測，首選乙腈-水系統(tǒng)）緩沖鹽：少用，盡可能低反相色譜系統(tǒng)，C-18柱，有機相比例應不低于5%。USP(32)High-purityreagentsand“HPLCgrade”organicsolventswater:lowcondutivityandlowUVabsorptionEP(6.0)Fornomal-phasechromatography,lesspolarsolventsareemployed.Wateristobestrictlycontrolled!0.45μmdegassedbysparingwithhelium,sonicationorusingon-linemembrane/vacuumAdjustmentofthepH,iseffectedusingonlytheaqueouscomponentofthemobilephaseandnotthemixture.2/4/202325山東省藥品檢驗所系統(tǒng)適用性SystemSuitability2/4/202326山東省藥品檢驗所定義CHP(2010):色譜系統(tǒng)的適用性試驗通常包括理論板數(shù)、分離度、重復性和拖尾因子四個參數(shù)，其中，分離度和重復性尤為重要。ICHdefinition:Systemsuitabilitytestingisanintegralpartofmanyanalyticalprocedures.Thetestsarebasedontheconceptthattheequipment,electronics,analyticaloperationsandsamplestobeanalyzedconstituteanintegralsystemthatcanbeevaluatedassuch.2/4/202327山東省藥品檢驗所DefinitionEP(6.0):Thesystemsuitabilitytestsrepresentanintegralpartofthemethodandareusedtoensureadequateperformanceofthechromatographicsystem.Thevariouscomponentsoftheequipmentemployedmustbequalifiedandbecapableofachievingtheprecisionrequiredtoconductthetestorassay.USP(32):

Systemsuitabilitytestsareanintegralpartofgasandliquidchromatographicmethods.Theyareusedtoverifythattheresolutionandreproducibilityofthechromatographicsystemareadequatefortheanalysistobedone.2/4/202328山東省藥品檢驗所DefinitionUSP(32):

Nosampleanalysisisacceptableunlesstherequirementsofsystemsuitablityhavebeenmet.Sampleanalysesobtainedwhilethesystemfailsrequirementsareunacceptable.Systemsuitabilitymustbedemonstratedthroughouttherunbyinjectionofanappropriatecontrolpreparationatappropriateintervals.Whereverthereisasignificantchangeinequipmentorinacriticalreagent,suitabilitytestingshouldbeperformedbeforetheinjectionofsamples.2/4/202329山東省藥品檢驗所理論板數(shù)評價色譜柱的重要指標。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：半高峰寬N=5.54×(tR/W0.5h)2W0.5h：半高峰寬影響因素：固定相、柱溫、流動相和保留時間。*有爭議時，以峰寬（W）計算結(jié)果為準2/4/202330山東省藥品檢驗所分離度（R）衡量色譜系統(tǒng)效能的關鍵指標！2/4/202331山東省藥品檢驗所分離度（R）CHP(2010)*R=2(tR2-tR1)/(W1+W2)R=2(tR2-tR1)/1.70(W1,h/2+W2,h/2)除另有規(guī)定外，應大于1.5

USP(32)*#

EP(6.0)#

JP(XV)*有爭議時，以峰寬（W）計算結(jié)果為準；#describedinindividualmonograph2/4/202332山東省藥品檢驗所USP(32)Chromatographicsystem—Theliquidchromatographisequippedwitha275-nmdetectoranda4.6-mm×15-cmcolumnthatcontainspackingL1……theresolution,

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

2/4/202333山東省藥品檢驗所EP(6.0)左甲狀腺素鈉含量測定：左羥丙哌嗪對映體純度：2/4/202334山東省藥品檢驗所重復性評價連續(xù)進樣中，色譜系統(tǒng)響應值的重復性能。CHP(2010):外標法：對照品溶液，連續(xù)進樣5次，峰面積RSD不得過2.0%。內(nèi)標法：配制相當于80%、100%、120%的對照品溶液，加入內(nèi)標溶液，分別至少進樣2次，計算平均校正因子的RSD不得過2.0%

。2/4/202335山東省藥品檢驗所重復性

USP(32):Unlessotherwisespecifiedintheindividualmonograph,datafrom

five

replicateinjectionsoftheanalyteareusedtocalculatetherelativestandarddeviation,SR,iftherequirmentis2.0%orless;datafromsix

replicateinjectionsareusediftherelativestandarddeviationrequirmentismorethan2.0%.

2/4/202336山東省藥品檢驗所重復性

EP(6.0):Therepeatabilityofresponseisexpressedasanes

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

standardisationmethodnumberofindividualvalues2/4/202337山東省藥品檢驗所重復性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山東省藥品檢驗所重復性NOTE:Thisrequirementdoesnotapplytotestsforrelatedsubstances.upperlimitgiveninthedefinitionoftheindividualmonographminus100%contant(0.349)numberofreplicateinjectionsofthereferencesolution(3≤n≤6)90%probabilitylevel，n-1degreesoffreedomEP(6.0):Inanassayofanactivesubstancewherethevalueis100percentforapuresubstance,themaximumpermitted(Sr(%)max)fordefinedlimitsiscalculatedusingthefollowingequation:2/4/202339山東省藥品檢驗所重復性

JP(XV):

公式與EP相同。重復次數(shù)及限度在各論中要求。例：

RitodrineHydrochlorideRelatedsubsances

Systemsuitability—Testforrequireddetectability:............Systemperformance:............Systemrepeatability:Whenthetestisrepeated6timeswith......，therelativestandarddeviationofthepeakareasofdeferoxamineisnotmorethan3.0%.2/4/202340山東省藥品檢驗所拖尾因子（對稱因子）用于評價色譜峰的對稱性2/4/202341山東省藥品檢驗所拖尾因子T（orSymmetryfactor）CHP(2010)T=W0.05h/2d1應符合個論項下的規(guī)定除另有規(guī)定外，峰高法定量時T應在0.95~1.05之間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山東省藥品檢驗所USPItisalsoacommonpracticetomeasuretheAsymmetryfactorastheratioofthedistancebetweentheverticallineconnectingthepeakapexwiththeinterpolatedbaselineandthepeakfront,andthedistancebetweenthatlineandthepeakbackmeasureat10%ofthepeakheight,itwouldbe(W0.10-f0.10)/f0.10However,forthepurposeofUSP,onlytheformulapresentedintheGlossaryofSymbolsisvalid.2/4/202343山東省藥品檢驗所EP中系統(tǒng)適用性：p/vandS/NThepeak-to-valleyratio(p/v)maybeemployedasasystemsuitabilitycriterioninatestforrelatedsubstanceswhenbaselineseparationbetween2peaksisnotachieved.p/v=Hp/HvForexample:Econazolerelatedsubstances2/4/202344山東省藥品檢驗所EPTheshort-termnoiseinfluencestheprecisionofquantification.The

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

Forexample:

ketotifenhydrogenfumarate

relatedsubstances2/4/202345山東省藥品檢驗所色譜條件的調(diào)整AdjustmentofChromatographicConditions2/4/202346山東省藥品檢驗所CHP(2010)明確規(guī)定：不可變的有：固定相的種類、流動相的組分、檢測器類型可變的有：色譜柱內(nèi)徑、長度、載體粒度、流動相流速、混合流動相各組分的比例、柱溫、進樣量、檢測器的靈敏度。2/4/202347山東省藥品檢驗所CHP(2010)與CHP(2005)不同：《中國藥典》2010年版規(guī)定了流動相調(diào)整的限度。調(diào)整流動相組分比例時，以組分比例較低者（≤50％）相對于自身改變量不超過±30％且相對于總量的改變量不超過±10％為限，如30％相對改變量的數(shù)值超過總量的10％時，則改變量以總量的±10％為限。2/4/202348山東省藥品檢驗所CHP(2010)例：奧美拉唑腸溶片，釋放度檢查，色譜條件：問題：峰型差！流動相可調(diào)范圍：82.5:17.5~67.5:32.5解決：70：302/4/202349山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所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山東省藥品檢驗所EP(6.0)Whencolumndimensionsarechanged,theflowratemaybeajustedasnecessaryusingequation:F1：規(guī)定的流速F2：調(diào)整后的流速l1：規(guī)定的柱長l2：實際所用的柱長d1：規(guī)定柱子的內(nèi)徑d2：實際所用柱子的內(nèi)徑2/4/202355山東省藥品檢驗所EP(6.0)pHoftheaqueouscomponentofthemobilephaseconcentrationofsaltsinbuffercompositionofthemobilephaseWavelengthofUVdetectorcolumntemperaturenoadjustmentpermitted

noadjustmentpermittedthesystemsuitabilityrequirementsarefulfilleldtheprincipalpeakelutewithin±15%oftheindicatedretentiontimeelutionpowerisnotweakerthanbeforenoadjustmentpermitted±5℃columnlengthinteraldiameterparticlesizeflowrateinjectionvolume±70%±25%noadjustmentpermittedsameasisocraticelutionmaybedecreasednoincreasepermittedGradientelution：morecriticalthanwithisocraticelution2/4/202356山東省藥品檢驗所HPLC的應用及方法開發(fā)2/4/202357山東省藥品檢驗所HPLC應用類型定

義I原料藥或藥物制劑活性成分中主要成分的分析程序-如含量測定II原料藥中雜質(zhì)檢查或藥物制劑中降解產(chǎn)物的分析程序，這些分析程序包括定量試驗或限度試驗III用于性能參數(shù)測試的分析程序-如溶出度、釋放度IV鑒別試驗HPLC可以用來干什么？2/4/202358山東省藥品檢驗所方法開發(fā)的指導原則ICHUSP《中國藥典》2/4/202359山東省藥品檢驗所ICHICH(人用藥品注冊技術要求國際協(xié)調(diào)會)三方協(xié)調(diào)指導原則

Q2A：分析方法論證的文本ICH三方協(xié)調(diào)指導原則

Q2B：方法學ICH三方協(xié)調(diào)指導原則2/4/202360山東省藥品檢驗所ICH分析方法的類型項目鑒別雜質(zhì)檢查含量分析，溶出度，含量/效價定量限度檢測準確