j第十章甾體激素類藥物的分析

TheAnalysisofSteroidHormoneDrugs甾體激素類藥物分析Chapter10Themaincontents：

甾體激素類藥物的基本結(jié)構(gòu)特點(diǎn)與分類

Thestructure,characteristicsandclassification；

甾體激素藥物的鑒別試驗(yàn)

Theidentificationtests；

甾體激素藥物的特殊雜質(zhì)檢查

Thespecificimpuritytests；

甾體激素藥物的含量測定

Assays.Fundamentalrequirement:

掌握各類甾體激素藥物的基本結(jié)構(gòu)特點(diǎn)、鑒別實(shí)驗(yàn)及含量測定方法；

Masteringthegeneralstructurecharacteristics、identificationtestsandassaysofsteroidhormones;

熟悉HPLC在鑒別試驗(yàn)及含量測定中的應(yīng)用；

FamiliarizingwiththeapplicationofHPLCinidentificationtestsandassays;

掌握IR在甾體激素類藥物鑒別中的應(yīng)用。

MasteringtheapplicationofIRinidentificationtests.AnalysisAnalysisStructureIdentificationAssaySteroidhormonesSpecificimpurityBriefsummaryTorequirementTocontent基本結(jié)構(gòu)與分類

結(jié)構(gòu)特點(diǎn)

基本骨架:

甾體激素類藥物，一些為天然物，一些為人工合成品。但無論是天然的還是人工合成的本類藥物，均含有環(huán)五烷駢多氫菲母核。多數(shù)藥物都是半合成的。第一節(jié)Generalstructures

Structurecharacteristics

Steroidskeleton:Steroidcompoundshaveacyclo-pentanoperhydrophenanthreneskeleton.Someofthesedrugsarenaturalproducts,andsomearesyntheticmaterials.Mostofthesedrugsareobtainedbysemi-syntheticmethods.Section1SortStructureTypicaldrugsAdrenocorticoidhormonesHydrocortisoneAcetate(醋酸氫化可的松）;DexamethasoneAcetate（醋酸地塞米松）;AndrogensMethyltestosterone（甲睪酮）;TestosteronePropinate（丙酸睪酮）;GalairanabolichormonesNandrolonePhenylpropionate(苯丙酸諾龍）;ProgestogensProgesterone;MegestrolAcetate（醋酸甲地孕酮）;Norethisterone（炔諾酮）;EstrogensEstradiol;Ethinylestradiol（炔雌醇）;Diethylstibestrol

(己烯雌酚）.HydrocortisoneAcetateDexamethasoneAcetateMethyltestosteroneTestosteronePropionateNandrolonePhenylpropionate苯丙酸諾龍Progesterone黃體酮MegestrolAcetate醋酸甲地孕酮Norethisterone炔諾酮Estradiol雌二醇Ethinylestradiol炔雌醇Diethylstibestrol己烯雌酚鑒別實(shí)驗(yàn)第二節(jié)Δ4-3-酮基—紫外分光光度法

C17-α-醇酮基—呈色反應(yīng)(與氧化劑四氮唑鹽呈色)—沉淀反應(yīng)酮基—呈色反應(yīng)(與羰基試劑呈色)—制備衍生物測定其熔點(diǎn)—與強(qiáng)酸的呈色反應(yīng)官能團(tuán)—紅外分光光度法甲基酮—呈色反應(yīng)(與亞硝基鐵氰化鈉、間二硝基酚、芳香醛呈色)IdentificationtestsSection2Δ4-3-keto—Ultravioletspectrophotometry

α-hydroxyacetone—Colorreaction(withtetrazolinesalt)—PrecipitationreactionKetonegroup—Colorreaction(withcarbonylreagents)—Meltingpointdeterminationofderivatives—ReactionwithstrongacidsFunctionalgroup—InfraredspectrophotometryMethylketonegroup—Colorreaction(withsodium

nitroferricyanide,m-dinitrophenol,aromaticaldehyde)鑒別實(shí)驗(yàn)Section2有機(jī)氟—呈色反應(yīng)(經(jīng)氧瓶燃燒后生成無機(jī)氟化物，再與茜素氟藍(lán)及硝酸亞鈰呈色）有機(jī)氯—沉淀反應(yīng)(經(jīng)有機(jī)破壞生成無機(jī)氯化物，在硝酸酸性下與硝酸銀作用，生成白色沉淀)酚羥基—呈色反應(yīng)

(與重氮苯磺酸反應(yīng)生成紅色偶氮染料)

—制備衍生物測定其熔點(diǎn)

酯基—水解產(chǎn)物的反應(yīng)炔基—沉淀反應(yīng)(與硝酸銀反應(yīng)生成白色沉淀)IdentificationtestsSection2Organicfluorine—Colorreaction(withalizarincomplexoneandCerousnitrateafterdestroyedbyoxygenflaskcombustion）Organicchlorine—Precipitationreaction(withnitricacidandsilvernitrateafterdestroyedbyoxygenflaskcombustion)

Phenolichydroxyl—Colorreaction(diazobenzenesulfonicacid)

—MeltingpointdeterminationofderivativesEstergroup—HydrolysisproductreactionAlkynylgroup—Precipitationreaction(withsilvernitrate)Names

ColorFluorescenceAddingwa-terdilutionCortisoneacetate

Yellowororange

-ColordisappearsandclearsolutionformedPrednisone

Orange-Yellowtoblue-greenReactionwithsulfuricacidUltravioletspectrophotometryConjugatedbondingsystems:1.α,β-unsaturatedketonegroup2.SteroidswitharomaticringATheUVspectraofhydrocotisoneandbetamethasonePrecipitationreactionReactionwithsilvernitratepreparedinammonia

Corticalhormones

SilvernitrateAmmoniaSilverprecipitate(black)Reactionwithsilvernitrate

Steroidhormones(alkynyl)SilvernitrateSilversaltprecipitate(white)MeltingpointsofderivativesTheformulationofketoxime（酮肟）+2HONH2+2H2O（Progesterone黃體酮）（Meltingpoint:235~240℃）MeltingpointofderivativesTheformulationofsemicarbazone（縮氨基脲）+

H2O+HCl（NandrolonePhenylpropionoate

苯丙酸諾龍）（Meltingpoint:180℃(dec.)）+

NH2CONHNH2·HClMeltingpointsofderivativesTheformulationofester++KOH+KCl+H2O（Meltingpoint:200-202℃）Ethinylestradiol炔雌醇Infraredspectrophotometry–OHstretching

everywhere~3600

aCH2&CH3stretchingeverywhere2970~2850

b=C–Hstretchingaromaticring3040~3010≡C–Hstretching~3320aliphaticketonestretchhexa-typering1720~1705cfive-membered1749~1742C20－1710~1706–OCOCH3

everywhere1742~1735–C=C–C=Ohexa-typering1684~1620d1585~1620(Δ4–3–keto)Assignmentlocationwavenumber(cm-1)comments–C–OH(alcohol)

everywhere1230~1000–C–OH(hydroxybenzene)

1300~1200–OCOR1200~1000–C=C–H

everywhere900~650Comments

a:Ifhydrogenbondswiththehydroxygroups,thebandshiftsto3550~3330cm-1.b:Oftenappearsasashoulder.c:C=Ostretching

inCCl4occursatalowwavenumber.d:AsaresultofhydrogenbondingwiththehydroxygroupstheνC=Obandofthe3-ketogroupsappearsatrelativelylowfrequency,andthustheνC=Obandcannotbeobserved.υC=C1615，1590，1505cm－1苯環(huán)的骨架振動(dòng)；υ≡CH3300cm－1

炔的特征峰；υ－OH3610cm－1游離酚羥基的伸縮振動(dòng)；υ－OH3505cm－1C17-羥基的伸縮振動(dòng)。

Ethinylestradiol

(炔雌醇)IRspectrumIRspectrumofDexamethasone(地塞米松)InfraredspectrophotometryPrinciples:

Electromagneticradiationrangingbetween500cm-1and4000cm-1(2500and20000nm)ispassedthroughasampleandisabsorbedbythebondsofthemoleculesinthesamplecausingthemtostretchorbend.Thewavelengthoftheradiationabsorbedischaracteristicofthebondabsorbingit.UnitTheunitofwavenumber(cm-1)isusedforIRSpectroscopy1,000μm0.4μm0.2μm0.7μm25μm2.5μm25,000cm-150,000cm-115,000cm-110cm-1400cm-14,000cm-1X-RayUVVISMicrowaveNIRFarIR

MidIRInfraredWavelengthWave

numberConversionWavenumber(cm-1)=10,000÷Wavelength(μm)Wavelength(μm)=10,000÷Wavenumber(cm-1)EnergyIncreaseWavelengthRange:0.7~1,000μm(15,000~10cm-1)InfraredRadiation

Theintensitywithwhichabondabsorbsradiationdependsonitsdipolemoment.Theorderofintensityofabsorptionis:

C–O>C–Cl>C–N>C–C–OH>C–C–HTheintensitydependsontherelativeelectronegativityoftheatomsinvolvedinthebond.Theintensityofthestretchingofcarbon-carbondoublebondsisincreasedwhentheyareconjugatedtoapolardoublebond.Theorderofintensityis:

C=C–C=O>C=C–C=C>C=C–C–FactorsdeterminingintensityinIRspectra:

TwotypesofinstrumentarecommonlyusedforobtainingIRspectra:dispersiveinstrumentswhichuseamonochromatortoselecteachwavenumberinturninordertomonitoritsintensityaftertheradiationhaspassedthroughthesampleandFouriertransforminstrumentsthatuseaninterferometer.

Instrumentation

ToSamplecompartmentToDetector０λ/2λ-λ/2-λFixedMirror(M2)MovingMirror(M1)SourceBeamsplitter(HalfMirror)InterferometerMichelsoninterferometerMichelsonInterferometerFixedmirrorMovingmirrorXBeamsplitterDetectorPolychromaticRadiationsource100%50%50%50%50%25%+25%25%+25%(Michelson:1890’s)Aqualitativefingerprintcheckfortheidentityofrawmaterialusedinmanufactureandforidentifyingdrugs.Usedinsyntheticchemistryasapreliminarycheckforcompoundidentityparticularlyforthepresenceorabsenceofacarbonylgroup,whichisdifficulttocheckbyanyothermethod.Canbeusedtocharacterizesamplesinthesolidandsemi-solidstatessuchascreamsandtablets.

Applications

Usedasafingerprinttestforfilms,coatingsandpackagingplastics.Canbeusedtodetectpolymorphsofdrugs(polymorphsaredifferentcrystalformsofamoleculethathavedifferentphysicalpropertiessuchassolubilityandmeltingpointwhichmaybeimportantinthemanufacturingprocess).

Applications

OilIndustryPharmaceuticalIndustryFoodIndustryIRForensicScienceMaterialsScienceBiologicalScienceIR............EnvironmentalSciencePolymersSemiconductorsCeramicFieldsofIRApplications

IRisoneofthemostlywidelyusedanalyticaltechniques.IRcanbeusedforbothqualitativeandquantitativeanalysisofanyphase.

Providesacomplexfingerprintwhichisuniquetothecompoundbeingexamined.Computercontrollfinstrumentsmeansthatmatchingofthespectrumofacompoundtoitsstandardfingerprintcannowbereadilycarriedout.

Strengths

RarelyusedasaquantitativetechniquebecauseofrelativedifficultyinsamplepreparationandthecomplexityIRspectra.Usuallycanonlydetectgrossimpuritiesinsamples.Samplepreparationrequiresadegreeofskill,particularlywhenpotassiumbromide(KBr)discsarebeingprepared.Thetechniqueislackinginrobustnesssincesamplehandlingcanhaveaneffectonthespectrumobtainedandthuscarehastobetakeninsampleprocessing.Limitations

特殊雜質(zhì)檢查

甾體激素藥物多由其它甾體化合物或結(jié)構(gòu)類似的其他甾體激素經(jīng)結(jié)構(gòu)改造而來，因而可能帶來原料、中間體、異構(gòu)體、降解產(chǎn)物以及試劑和溶劑等雜質(zhì)。甾體激素類藥物在純度檢查時(shí)，除一般雜質(zhì)外，檢查其特殊“其他甾體”的限度，是一個(gè)重要的項(xiàng)目。

第三節(jié)TheSpecificImpuritytests

Steroidhormonedrugsareobtainedbymodifyingothersteroidcompoundsorotherstructurallyrelativesteroidhormones.Impurities,suchasrawmaterials、intermediatecompounds、isomers、degradationproducts、reagentsandsolventsetc,maybeintroducedintotheirfinalproducts.Exceptforgeneralimpurities,thelimitofothersteroidsisalsoanimportantiteminthepuritytests.Section3地塞米松磷酸鈉、氫化可的松磷酸鈉——游離磷酸——測吸收度地塞米松磷酸鈉——甲醇和丙酮——?dú)庀嗌V法炔雌醇——雌酮——比色法醋酸地塞米松、醋酸氟輕松——硒——氧甁法-比色法甾體激素——其它甾體——薄層色譜法、高效液相色譜法舉例Dexamethasonesodiumphosphate、hydrocortisonesodiumphosphate——freephosphonicacid——determinationofADexamethasonesodiumphosphate——methanolandacetone——GCEthinyloestradiol——estrone——colorimetryDexamethasoneacetate、fluocinonideacetate——selenium——OCF-colorimetrySteroidhormones——othersteroids——TLC、HPLCExamples四氮唑比色法（Tetrazolinecolorimetry）異煙肼比色法（Isoniazidecolorimetry）Kober反應(yīng)比色法（Kober

reactioncolorimetry)紫外分光光度法(UV)高效液相色譜法(HPLC)AssaysSection4四氮唑比色法常用的四氮唑鹽：2,3,5-三苯基氯化四氮唑，其還原產(chǎn)物為不溶于的深紅色的三苯甲，也稱紅四氮唑(RT)；3,3’-二甲氧苯基-雙-4,4’-(3,5-二苯基)氯化四氮唑,其還原產(chǎn)物為暗藍(lán)色的雙甲，也稱藍(lán)四氮唑。Tetrazolinecolorimetry

Commonlyusedtetrazolinesalts：Redtetrazoline(RT):2,3,5-triphenyltetrazoliumchloride(TTC);Bluetetrazoline(BT):{3,3’-dianisole-bis[4,4’-(3,5-diphenyl)tetrazoliumchloride]}TTCBT

Principle：皮質(zhì)激素C

17

-α

-醇酮基具有還原性，在強(qiáng)堿性溶液中能將四氮唑鹽定量的還原為有色甲(formazan)。Method：USP(24)采用藍(lán)四氮唑；BP(1998)采用氯化三苯四氮唑；中國藥典(2000)也采用氯化三苯四氮唑試劑。2e以中國藥典（2000年版）收載的醋酸潑尼松龍軟膏的含量測定為例，測定方法如下：

精密稱取對照品20mg加無水乙醇100ml量瓶中振搖溶解稀釋至刻度搖勻?qū)φ掌啡芤壕芊Q取樣品4g加無水乙醇約30ml燒杯中水浴加熱攪拌溶解冰浴冷卻濾過濾液加無水乙醇稀釋搖勻供試品溶液供試品溶液對照品溶液各精密量取1ml加無水乙醇9ml氯化三苯四氮唑液2ml氫氧化四甲基銨試液1ml搖勻放置40～45min25℃測A計(jì)算即得影響因素：

A.

基團(tuán)影響：反應(yīng)速度C11-酮基>C11-羥基；C21-羥基>C21-羥基酯。B.

溶劑和水分影響：含水量大時(shí)會(huì)使呈色速度減慢，故采用無水乙醇；醛會(huì)使吸收度增高，故采用無醛醇。堿的影響：最常用氫氧化四甲基銨，能得到滿意結(jié)果。為防止皮質(zhì)激素與堿長時(shí)接觸部分分解，以先加入四氮唑鹽再加堿液較好。空氣中氧及光線的影響：反應(yīng)及其產(chǎn)物對光敏感，因此必須用避光容器并置于暗處顯色，同時(shí)在達(dá)到最大呈色時(shí)間后，立即測定吸收度。E.

溫度和時(shí)間的影響：呈色速度隨溫度增高而加快。一般以室溫或30℃恒溫條件下顯色，易得重現(xiàn)性較好的結(jié)果。

影響因素：

Influencefactors：

A.

FunctionalgroupsTheyofteninfluencetherateofreaction:C11-keto>C11-hydroxy；C21-hydroxy>C21-hydroxylester。B.

SolventandmoistureWaterwillslowdowntherateofcolorreaction,weusenon-aqueousethanolasthesolventinsteadofwater.Asaldehydewillmaketheabsorbancehigh,weusenon-aldehydeethanol.AlkalinereagentTetramethylammoniumhydroxideiscommonlyusedforasatisfiedresult.Notice:addtetrazolinesaltfirstandthenalkalinesolutiontoprohibitthedecompositionofcorticosteroids.OxygenandlightBecausereactionproductsaresensitivetotheray,photophobiccontainerisneededandplacedinthedark.Immediatedeterminationisneeded.E.TemperatureandtimeHigherthetemperatureis,fastertherateofcolorreactionis.Generally,roomtemperatureor30℃constanttemperatureisthebestcondition.Theresulthasabetterrepeatability.Influencefactors：

原理：甾體激素C3-酮基及某些其他位置上的酮基都能在酸性條件下與羰基試劑異煙肼縮和形成黃色異煙腙，在一定波長下具有最大吸收。

Isoniazidcolorimetricmethod++H2O本法主要用于甾體激素制劑的測定。KoberReactionColorimetry：

Koberreactionisacolorreactionofestrogenandsulfate-ethanolwhenheating.Thecolorwillconverttoberedwhendilutingwithwaterordilutedsulfuricacidorheatingagain.Themaximumabsorptionisat515nm.Thisreactionhastwosteps：first:estrogenisheatedtogetherwithsulfuricacid-ethanoltoappearyellowcolor,whichhasthemaximumabsorptionaround465nm;secondly:thesolutionappearspinkwhendilutingwithwaterordilutedsulfuricacidorheatingagain.Themaximumabsorptionisat515nm.

具有甾體母核；

A環(huán)為芳環(huán)；C3-為-OH,或?yàn)?OCH3，或?yàn)?OCOCH3； C17-OH; C13為角甲基。Requirements鐵-柯柏試劑(Iron-Koberreagent)。

硫酸亞鐵銨加水溶解后，加硫酸及過氧化氫，再與苯酚混合制成。鐵酚試劑比色法具有以下優(yōu)點(diǎn)：由于加入少量鐵鹽，能加速黃色形成的速率和強(qiáng)度；加速黃色轉(zhuǎn)變?yōu)榧t色，也能增強(qiáng)紅色的穩(wěn)定性；酚的加入可以消除反應(yīng)中產(chǎn)生的熒光并加速紅色的形成。鐵酚試劑(Iron-phenolReagent)UltravioletSpectroscopy原理:許多甾體激素分子中存在酮和苯環(huán)共軛體系，因而在紫外光區(qū)有特征吸收具有酮基結(jié)構(gòu)的皮質(zhì)激素、雄性激素、孕激素以及許多口服避孕藥，在240nm處有最大吸收。具有苯環(huán)的的雌激素在280nm附近有最大吸收。這些吸收特征都可用于含量測定。

Principle:ManysteroidhormoneshavecharacteristicUVabsorptionbecauseofdoublebonds(aromaticringA)andtheirconjugationwithketogroupsonthem.WiththestructureofΔ4-3-keto

corticoidhormones,androgens,progestogensandmanyperoralcontraceptiveshavethemaximumabsorptionatabout240nm.Withthestructureofbenzoicring,estrogenshavethemaximumabsorptionatabout280nm.高效液相色譜法高效液相色譜法具有樣品用量少、靈敏度高、分離效能好、快速等優(yōu)點(diǎn)，因此被各國藥典USP(25),BP(2000)、JP(14)采用。HPLC在本類藥物的含量測定中，占到分析方法的48%，居各種分析方法之首。在高效液相色譜法中，以反相分配色譜用于甾體激素的含量測定最為廣泛。固定相常用十八烷基硅烷鍵合硅膠；流動(dòng)相大都是甲醇和水組成的混合液。為了提高分離效果，有時(shí)在流動(dòng)相中加入醋酸緩沖液或磷酸緩沖液調(diào)節(jié)流動(dòng)相的pH值。許多甾體激素都具有Δ4-3-酮基和苯環(huán)，直接采取紫外檢測器（240nm或280nm波長附近）檢測。HPLC

Advantage