j第十章甾体激素类药物的分析

TheAnalysisofSteroidHormoneDrugs甾体激素类药物分析Chapter10Themaincontents：

甾体激素类药物的基本结构特点与分类

Thestructure,characteristicsandclassification；

甾体激素药物的鉴别试验

Theidentificationtests；

甾体激素药物的特殊杂质检查

Thespecificimpuritytests；

甾体激素药物的含量测定

Assays.Fundamentalrequirement:

掌握各类甾体激素药物的基本结构特点、鉴别实验及含量测定方法；

Masteringthegeneralstructurecharacteristics、identificationtestsandassaysofsteroidhormones;

熟悉HPLC在鉴别试验及含量测定中的应用；

FamiliarizingwiththeapplicationofHPLCinidentificationtestsandassays;

掌握IR在甾体激素类药物鉴别中的应用。

MasteringtheapplicationofIRinidentificationtests.AnalysisAnalysisStructureIdentificationAssaySteroidhormonesSpecificimpurityBriefsummaryTorequirementTocontent基本结构与分类

结构特点

基本骨架:

甾体激素类药物，一些为天然物，一些为人工合成品。但无论是天然的还是人工合成的本类药物，均含有环五烷骈多氢菲母核。多数药物都是半合成的。第一节Generalstructures

Structurecharacteristics

Steroidskeleton:Steroidcompoundshaveacyclo-pentanoperhydrophenanthreneskeleton.Someofthesedrugsarenaturalproducts,andsomearesyntheticmaterials.Mostofthesedrugsareobtainedbysemi-syntheticmethods.Section1SortStructureTypicaldrugsAdrenocorticoidhormonesHydrocortisoneAcetate(醋酸氢化可的松）;DexamethasoneAcetate（醋酸地塞米松）;AndrogensMethyltestosterone（甲睾酮）;TestosteronePropinate（丙酸睾酮）;GalairanabolichormonesNandrolonePhenylpropionate(苯丙酸诺龙）;ProgestogensProgesterone;MegestrolAcetate（醋酸甲地孕酮）;Norethisterone（炔诺酮）;EstrogensEstradiol;Ethinylestradiol（炔雌醇）;Diethylstibestrol

(己烯雌酚）.HydrocortisoneAcetateDexamethasoneAcetateMethyltestosteroneTestosteronePropionateNandrolonePhenylpropionate苯丙酸诺龙Progesterone黄体酮MegestrolAcetate醋酸甲地孕酮Norethisterone炔诺酮Estradiol雌二醇Ethinylestradiol炔雌醇Diethylstibestrol己烯雌酚鉴别实验第二节Δ4-3-酮基—紫外分光光度法

C17-α-醇酮基—呈色反应(与氧化剂四氮唑盐呈色)—沉淀反应酮基—呈色反应(与羰基试剂呈色)—制备衍生物测定其熔点—与强酸的呈色反应官能团—红外分光光度法甲基酮—呈色反应(与亚硝基铁氰化钠、间二硝基酚、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)鉴别实验Section2有机氟—呈色反应(经氧瓶燃烧后生成无机氟化物，再与茜素氟蓝及硝酸亚铈呈色）有机氯—沉淀反应(经有机破坏生成无机氯化物，在硝酸酸性下与硝酸银作用，生成白色沉淀)酚羟基—呈色反应

(与重氮苯磺酸反应生成红色偶氮染料)

—制备衍生物测定其熔点

酯基—水解产物的反应炔基—沉淀反应(与硝酸银反应生成白色沉淀)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黄体酮）（Meltingpoint:235~240℃）MeltingpointofderivativesTheformulationofsemicarbazone（缩氨基脲）+

H2O+HCl（NandrolonePhenylpropionoate

苯丙酸诺龙）（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苯环的骨架振动；υ≡CH3300cm－1

炔的特征峰；υ－OH3610cm－1游离酚羟基的伸缩振动；υ－OH3505cm－1C17-羟基的伸缩振动。

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

特殊杂质检查

甾体激素药物多由其它甾体化合物或结构类似的其他甾体激素经结构改造而来，因而可能带来原料、中间体、异构体、降解产物以及试剂和溶剂等杂质。甾体激素类药物在纯度检查时，除一般杂质外，检查其特殊“其他甾体”的限度，是一个重要的项目。

第三节TheSpecificImpuritytests

Steroidhormonedrugsareobtainedbymodifyingothersteroidcompoundsorotherstructurallyrelativesteroidhormones.Impurities,suchasrawmaterials、intermediatecompounds、isomers、degradationproducts、reagentsandsolventsetc,maybeintroducedintotheirfinalproducts.Exceptforgeneralimpurities,thelimitofothersteroidsisalsoanimportantiteminthepuritytests.Section3地塞米松磷酸钠、氢化可的松磷酸钠——游离磷酸——测吸收度地塞米松磷酸钠——甲醇和丙酮——气相色谱法炔雌醇——雌酮——比色法醋酸地塞米松、醋酸氟轻松——硒——氧甁法-比色法甾体激素——其它甾体——薄层色谱法、高效液相色谱法举例Dexamethasonesodiumphosphate、hydrocortisonesodiumphosphate——freephosphonicacid——determinationofADexamethasonesodiumphosphate——methanolandacetone——GCEthinyloestradiol——estrone——colorimetryDexamethasoneacetate、fluocinonideacetate——selenium——OCF-colorimetrySteroidhormones——othersteroids——TLC、HPLCExamples四氮唑比色法（Tetrazolinecolorimetry）异烟肼比色法（Isoniazidecolorimetry）Kober反应比色法（Kober

reactioncolorimetry)紫外分光光度法(UV)高效液相色谱法(HPLC)AssaysSection4四氮唑比色法常用的四氮唑盐：2,3,5-三苯基氯化四氮唑，其还原产物为不溶于的深红色的三苯甲，也称红四氮唑(RT)；3,3’-二甲氧苯基-双-4,4’-(3,5-二苯基)氯化四氮唑,其还原产物为暗蓝色的双甲，也称蓝四氮唑。Tetrazolinecolorimetry

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

Principle：皮质激素C

17

-α

-醇酮基具有还原性，在强碱性溶液中能将四氮唑盐定量的还原为有色甲(formazan)。Method：USP(24)采用蓝四氮唑；BP(1998)采用氯化三苯四氮唑；中国药典(2000)也采用氯化三苯四氮唑试剂。2e以中国药典（2000年版）收载的醋酸泼尼松龙软膏的含量测定为例，测定方法如下：

精密称取对照品20mg加无水乙醇100ml量瓶中振摇溶解稀释至刻度摇匀对照品溶液精密称取样品4g加无水乙醇约30ml烧杯中水浴加热搅拌溶解冰浴冷却滤过滤液加无水乙醇稀释摇匀供试品溶液供试品溶液对照品溶液各精密量取1ml加无水乙醇9ml氯化三苯四氮唑液2ml氢氧化四甲基铵试液1ml摇匀放置40～45min25℃测A计算即得影响因素：

A.

基团影响：反应速度C11-酮基>C11-羟基；C21-羟基>C21-羟基酯。B.

溶剂和水分影响：含水量大时会使呈色速度减慢，故采用无水乙醇；醛会使吸收度增高，故采用无醛醇。碱的影响：最常用氢氧化四甲基铵，能得到满意结果。为防止皮质激素与碱长时接触部分分解，以先加入四氮唑盐再加碱液较好。空气中氧及光线的影响：反应及其产物对光敏感，因此必须用避光容器并置于暗处显色，同时在达到最大呈色时间后，立即测定吸收度。E.

温度和时间的影响：呈色速度随温度增高而加快。一般以室温或30℃恒温条件下显色，易得重现性较好的结果。

影响因素：

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：甾体激素C3-酮基及某些其他位置上的酮基都能在酸性条件下与羰基试剂异烟肼缩和形成黄色异烟腙，在一定波长下具有最大吸收。

Isoniazidcolorimetricmethod++H2O本法主要用于甾体激素制剂的测定。KoberReactionColorimetry：

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

具有甾体母核；

A环为芳环；C3-为-OH,或为-OCH3，或为-OCOCH3； C17-OH; C13为角甲基。Requirements铁-柯柏试剂(Iron-Koberreagent)。

硫酸亚铁铵加水溶解后，加硫酸及过氧化氢，再与苯酚混合制成。铁酚试剂比色法具有以下优点：由于加入少量铁盐，能加速黄色形成的速率和强度；加速黄色转变为红色，也能增强红色的稳定性；酚的加入可以消除反应中产生的荧光并加速红色的形成。铁酚试剂(Iron-phenolReagent)UltravioletSpectroscopy原理:许多甾体激素分子中存在酮和苯环共轭体系，因而在紫外光区有特征吸收具有酮基结构的皮质激素、雄性激素、孕激素以及许多口服避孕药，在240nm处有最大吸收。具有苯环的的雌激素在280nm附近有最大吸收。这些吸收特征都可用于含量测定。

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

corticoidhormones,androgens,progestogensandmanyperoralcontraceptiveshavethemaximumabsorptionatabout240nm.Withthestructureofbenzoicring,estrogenshavethemaximumabsorptionatabout280nm.高效液相色谱法高效液相色谱法具有样品用量少、灵敏度高、分离效能好、快速等优点，因此被各国药典USP(25),BP(2000)、JP(14)采用。HPLC在本类药物的含量测定中，占到分析方法的48%，居各种分析方法之首。在高效液相色谱法中，以反相分配色谱用于甾体激素的含量测定最为广泛。固定相常用十八烷基硅烷键合硅胶；流动相大都是甲醇和水组成的混合液。为了提高分离效果，有时在流动相中加入醋酸缓冲液或磷酸缓冲液调节流动相的pH值。许多甾体激素都具有Δ4-3-酮基和苯环，直接采取紫外检测器（240nm或280nm波长附近）检测。HPLC

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