j第十章甾体激素类药物分析

1、j第十章甾体激素类药物分析Fundamental requirement: 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； Mastering the general structure characteristics、identification tests and assays of steroid hormones; 熟悉HPLC在鉴别试验及含量测定中的应用； Familiarizing with the application of HPLC in identification tests and assays; 掌握IR在甾体激素类药物鉴别中的应用。 Masterin

2、g the application of IR in identification tests.AnalysisAnalysisStructureIdentificationAssaySteroid hormonesSpecific impurityBrief summaryTo requirementTo content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General structures Structure characteristics Ster

3、oid skeleton:Steroid compounds have a cyclo- pentanoperhydrophenanthrene skeleton.Some of these drugs are natural products,and some are synthetic materials.Most of these drugs are obtained by semi-synthetic methods.Section 1SortStructureTypical drugsAdrenocorticoid hormonesHydrocortisone Acetate(醋酸氢

4、化可的松）;Dexamethasone Acetate（醋酸地塞米松）;AndrogensMethyltestosterone（甲睾酮）;Testosterone Propinate（丙酸睾酮）;Galair anabolic hormonesNandrolone Phenylpropionate(苯丙酸诺龙）;ProgestogensProgesterone;Megestrol Acetate（醋酸甲地孕酮）;Norethisterone（炔诺酮）;EstrogensEstradiol;Ethinylestradiol（炔雌醇）;Diethylstibestrol (己烯雌酚）.Hydroc

5、ortisone AcetateDexamethasone AcetateMethyltestosteroneTestosterone PropionateNandrolone Phenylpropionate 苯丙酸诺龙Progesterone 黄体酮Megestrol Acetate 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4-3-酮基 紫外分光光度法 C17-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

6、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 )Identification testsSection 24-3-keto Ultraviolet spectrophotometry -hydroxyacetone Color reaction(with tetrazoline salt) Precipitation reactionKetone group Color reaction (with carbonyl reagents) Melting point determination of derivatives

7、Reaction with strong acidsFunctional group Infrared spectrophotometryMethylketone group Color reaction(with sodium nitroferricyanide , m-dinitrophenol,aromatic aldehyde )鉴 别 实 验Section 2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

8、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)Identification testsSection 2Organic fluorine Color reaction(with alizarin complexone and Cerous nitrate after destroyed by oxygen flask combustion ） Organic chlorine Precipitation reaction(with nitric acid and silver nitrate after destroyed by oxygen flask com

9、bustion) Phenolic hydroxyl Color reaction (diazobenzenesulfonic acid) Melting point determination of derivativesEster group Hydrolysis product reactionAlkynyl group Precipitation reaction(with silver nitrate)Names Color Fluorescence Adding wa- ter dilution Cortisone acetate Yellow or orange -Color d

10、isappears and clear solution formedPrednisone Orange -Yellow to blue-green Reaction with sulfuric acidUltraviolet spectrophotometryConjugated bonding systems: 1.,-unsaturated ketone group 2. Steroids with aromatic ring AThe UV spectra of hydrocotisone and betamethasonePrecipitation reactionReaction

11、with silver nitrate prepared in ammonia Cortical hormones Silver nitrateAmmonia Silver precipitate( black )Reaction with silver nitrate Steroid hormones( alkynyl )Silver nitrate Silver salt precipitate( white )Melting points of derivativesThe formulation of ketoxime（酮肟）+ 2HONH2+ 2H2O（Progesterone 黄体

12、酮）（Melting point: 235240）Melting point of derivativesThe formulation of semicarbazone（缩氨基脲）+ H2O + HCl（Nandrolone Phenylpropionoate 苯丙酸诺龙）（Melting point: 180(dec.)）+ NH2CONHNH2 HClMelting points of derivativesThe formulation of ester+ KOH+ KCl + H2O（Melting point: 200-202）Ethinylestradiol 炔雌醇Infrare

13、d spectrophotometryOH stretching everywhere 3600 a CH2 &CH3 stretching everywhere 29702850 b =CH stretching aromatic ring 30403010CH stretching 3320 aliphatic ketone stretch hexa-type ring 17201705 c five-membered 17491742 C20 17101706 OCOCH3 everywhere 17421735 C=CC=O hexa-type ring 16841620 d 1585

14、1620 (43 keto)Assignment location wavenumber (cm-1) commentsCOH (alcohol ) everywhere 12301000 COH (hydroxybenzene) 13001200 OCOR 12001000C=CH everywhere 900650Comments a: If hydrogen bonds with the hydroxy groups, the band shifts to 35503330cm-1. b: Often appears as a shoulder. c: C=O stretching in

15、 CCl4 occurs at a low wavenumber. d: As a result of hydrogen bonding with the hydroxy groups the C=O band of the 3-keto groups appears at relatively low frequency, and thus theC=O band cannot be observed. C=C1615，1590，1505cm1苯环的骨架振动；CH 3300cm1 炔的特征峰；OH 3610cm1游离酚羟基的伸缩振动；OH 3505cm1 C17- 羟基的伸缩振动。 Ethi

16、nylestradiol (炔雌醇) IR spectrumIR spectrum of Dexamethasone(地塞米松)Infrared spectrophotometryPrinciples : Electromagnetic radiation ranging between 500 cm-1 and 4000 cm-1(2500 and 20000 nm)is passed through a sample and is absorbed by the bonds of the molecules in the sample causing them to stretch or

17、bend. The wavelength of the radiation absorbed is characteristic of the bond absorbing it. UnitThe unit of wave number(cm-1) is used for IR Spectroscopy1,000m0.4m0.2m0.7m25m2.5m25,000 cm-1 50,000 cm-115,000 cm-110 cm-1 400 cm-1 4,000 cm-1X-RayUVVISMicrowaveNIRFar IR Mid IRInfrared WavelengthWave num

18、berConversionWave number (cm-1)=10,000 Wavelength (m)Wavelength (m)=10,000 Wave number (cm-1)Energy IncreaseWavelength Range :0.71,000m(15,00010cm-1)Infrared Radiation The intensity with which a bond absorbs radiation depends on its dipole moment. The order of intensity of absorption is: CO C Cl C N

19、 C COH CCH The intensity depends on the relative electronegativity of the atoms involved in the bond. The intensity of the stretching of carbon-carbon double bonds is increased when they are conjugated to a polar double bond. The order of intensity is : C=CC=O C=CC=C C=CCFactors determining intensit

20、y in IR spectra: Two types of instrument are commonly used for obtaining IR spectra: dispersive instruments which use a monochromator to select each wavenumber in turn in order to monitor its intensity after the radiation has passed through the sample and Fourier transform instruments that use an in

21、terferometer. Instrumentation To Sample compartmentTo Detector/2-/2-Fixed Mirror(M2)Moving Mirror (M1)SourceBeam splitter(Half Mirror)InterferometerMichelson interferometerMichelson InterferometerFixed mirrorMoving mirrorXBeamsplitterDetectorPolychromaticRadiation source100%50%50%50%50%25% + 25%25%+

22、 25%(Michelson: 1890s)A qualitative fingerprint check for the identity of raw material used in manufacture and for identifying drugs. Used in synthetic chemistry as a preliminary check for compound identity particularly for the presence or absence of a carbonyl group, which is difficult to check by

23、any other method. Can be used to characterize samples in the solid and semi-solid states such as creams and tablets. Applications Used as a fingerprint test for films, coatings and packaging plastics. Can be used to detect polymorphs of drugs (polymorphs are different crystal forms of a molecule tha

24、t have different physical properties such as solubility and melting point which may be important in the manufacturing process). Applications Oil IndustryPharmaceutical IndustryFood IndustryIRForensic ScienceMaterialsScienceBiological ScienceIR . .EnvironmentalSciencePolymersSemiconductorsCeramicFiel

25、ds of IR Applications IR is one of the mostly widely used analytical techniques. IR can be used for both qualitative and quantitative analysis of any phase. Provides a complex fingerprint which is unique to the compound being examined. Computer control lf instruments means that matching of the spect

26、rum of a compound to its standard fingerprint can now be readily carried out. Strengths Rarely used as a quantitative technique because of relative difficulty in sample preparation and the complexity IR spectra. Usually can only detect gross impurities in samples. Sample preparation requires a degre

27、e of skill, particularly when potassium bromide (KBr) discs are being prepared. The technique is lacking in robustness since sample handling can have an effect on the spectrum obtained and thus care has to be taken in sample processing. 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、

28、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The Specific Impurity tests Steroid hormone drugs are obtained by modifying other steroid compounds or other structurally relative steroid hormones. Impurities,such as raw materials、intermediate compounds、isomers、degradation pro

29、ducts 、 reagents and solvents etc, may be introduced into their final products. Except for general impurities, the limit of other steroids is also an important item in the purity tests.Section 3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Dexamethasone

30、 sodium phosphate、hydrocortisone sodium phosphatefree phosphonic acid determination of ADexamethasone sodium phosphatemethanol and acetoneGCEthinyloestradiol estrone colorimetryDexamethasone acetate、fluocinonide acetateseleniumOCF-colorimetrySteroid hormonesother steroidsTLC、HPLCExamples四氮唑比色法（ Tetr

31、azoline colorimetry）异烟肼比色法（Isoniazide colorimetry）Kober反应比色法（Kober reaction colorimetry)紫外分光光度法(UV)高效液相色谱法(HPLC)AssaysSection 4四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。Tetrazoline colorimetry Commonly used tetrazoline salts： Red

32、 tetrazoline(RT) : 2,3,5-triphenyltetrazolium chloride (TTC); Blue tetrazoline(BT): 3,3-dianisole-bis4,4-(3,5-diphenyl) tetrazolium chlorideTTCBT Principle： 皮质激素C 17 - -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

33、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4045min25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

34、。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： Influence factors： A.Functional groups They often influence the rate of reaction : C11-ketoC11-hydroxy；C21-hydroxyC21-hydroxyl ester。B. Solvent an

35、d moisture Water will slow down the rate of color reaction, we use non-aqueous ethanol as the solvent instead of water. As aldehyde will make the absorbance high, we use non-aldehyde ethanol. Alkaline reagent Tetramethyl ammonium hydroxide is commonly used for a satisfied result. Notice: add tetrazo

36、line salt first and then alkaline solution to prohibit the decomposition of corticosteroids.Oxygen and light Because reaction products are sensitive to the ray, photophobic container is needed and placed in the dark. Immediate determinationis needed.E. Temperature and time Higher the temperature is

37、, faster the rate of color reaction is. Generally, room temperature or 30 constant temperature is the best condition. The result has a better repeatability.Influence factors： 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。 Isoniazid colorimetric method+ H2O本法主要用于甾体激素制剂的测定。Kober React

38、ion Colorimetry： Kober reaction is a color reaction of estrogen and sulfate-ethanol when heating.The color will convert to be red when diluting with water or diluted sulfuric acid or heating again.The maximum absorption is at 515nm. This reaction has two steps：first: estrogen is heated together with

39、 sulfuric acid- ethanol to appear yellow color, which has the maximum absorption around 465nm; secondly:the solution appears pink when diluting with water or diluted sulfuric acid or heating again.The maximum absorption is at 515nm.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

40、铁-柯柏试剂(Iron-Kober reagent)。 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-phenol Reagent)Ultraviolet Spectroscopy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: Many steroid hormones have characteristic UV absorption because of double bonds (aromatic ring A) and their conjugation with keto groups on them. With the structure of 4-3-keto corticoid hormones, androgens, progestogens and many