The Analysis of Steroid Hormone Drugs

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1 The Analysis of Steroid Hormone Drugs
Chapter 10 甾体激素类药物分析

2 The main contents： 甾体激素类药物的基本结构特点与分类
The structure, characteristics and classification； 甾体激素药物的鉴别试验 The identification tests； 甾体激素药物的特殊杂质检查 The specific impurity tests； 甾体激素药物的含量测定 Assays.

3 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。 Mastering the application of IR in identification tests.

4 Analysis Analysis To content To requirement Steroid hormones
Specific impurity Steroid hormones Brief summary Structure Identification Assay

5 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

6 General structures Section 1 Structure characteristics
Steroid 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 1

7 Adrenocorticoid hormones
Sort Structure Typical drugs Adrenocorticoid hormones Hydrocortisone Acetate (醋酸氢化可的松）; Dexamethasone Acetate （醋酸地塞米松）; Androgens Methyltestosterone （甲睾酮）; Testosterone Propinate （丙酸睾酮）;

8 Galair anabolic hormones
Nandrolone Phenylpropionate (苯丙酸诺龙）; Progestogens Progesterone; Megestrol Acetate （醋酸甲地孕酮）; Norethisterone （炔诺酮）; Estrogens Estradiol; Ethinylestradiol （炔雌醇）; Diethylstibestrol (己烯雌酚）.

9 Hydrocortisone Acetate
Dexamethasone Acetate Methyltestosterone Testosterone Propionate

10 Nandrolone Phenylpropionate 苯丙酸诺龙
Progesterone 黄体酮 Megestrol Acetate 醋酸甲地孕酮

11 Diethylstibestrol 己烯雌酚
Estradiol 雌二醇 Norethisterone 炔诺酮 Ethinylestradiol 炔雌醇

12 鉴 别 实 验 Δ4-3-酮基 — 紫外分光光度法 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 C17-α-醇酮基 — 呈色反应 (与氧化剂四氮唑盐呈色) — 沉淀反应 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 ) 第二节

13 Identification tests Δ4-3-keto — Ultraviolet spectrophotometry
Ketone group — Color reaction (with carbonyl reagents) — Melting point determination of derivatives — Reaction with strong acids Functional group — Infrared spectrophotometry α-hydroxyacetone — Color reaction(with tetrazoline salt) — Precipitation reaction Methylketone group — Color reaction(with sodium nitroferricyanide , m-dinitrophenol,aromatic aldehyde ) Section 2

14 鉴 别 实 验 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 炔基 — 沉淀反应 (与硝酸银反应生成白色沉淀) 酯基 — 水解产物的反应 Section 2

15 Identification tests Organic 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 combustion) Phenolic hydroxyl — Color reaction (diazobenzenesulfonic acid) — Melting point determination of derivatives Alkynyl group — Precipitation reaction(with silver nitrate) Ester group — Hydrolysis product reaction Section 2

16 Adding wa- ter dilution Color disappears and clear solution formed
Reaction with sulfuric acid Names Color Fluorescence Adding wa- ter dilution Cortisone acetate Yellow or orange - Color disappears and clear solution formed Prednisone Orange Yellow to blue-green

17 Ultraviolet spectrophotometry
Conjugated bonding systems: 1.α,β-unsaturated ketone group 2. Steroids with aromatic ring A

18 The UV spectra of hydrocotisone and betamethasone

19 Silver salt precipitate
Precipitation reaction Reaction with silver nitrate prepared in ammonia Cortical hormones Silver nitrate Silver precipitate ( black ) Ammonia Reaction with silver nitrate Steroid hormones ( alkynyl ) Silver nitrate Silver salt precipitate ( white )

20 Melting points of derivatives
The formulation of ketoxime（酮肟） + 2HONH2 + 2H2O （Progesterone 黄体酮） （Melting point: 235~240℃）

21 Melting point of derivatives
The formulation of semicarbazone（缩氨基脲） + NH2CONHNH2 · HCl （Nandrolone Phenylpropionoate 苯丙酸诺龙） + H2O + HCl （Melting point: 180℃(dec.)）

22 Melting points of derivatives
The formulation of ester + + KOH Ethinylestradiol 炔雌醇 + KCl + H2O （Melting point: ℃）

23 Infrared spectrophotometry
Assignment location wavenumber (cm-1) comments –OH stretching everywhere ~ a CH2 &CH3 stretching everywhere ~ b =C–H stretching aromatic ring ~3010 ≡C–H stretching ~3320 aliphatic ketone stretch hexa-type ring 1720~ c five-membered 1749~1742 C20－ ~1706 –OCOCH everywhere ~1735 –C=C–C=O hexa-type ring 1684~ d (Δ4–3 – keto) 1585~1620

24 –C–OH (alcohol ) everywhere 1230~1000
–C–OH (hydroxybenzene) ~1200 –OCOR ~1000 –C=C–H everywhere ~650 Comments a: If hydrogen bonds with the hydroxy groups, the band shifts to 3550~3330cm-1. b: Often appears as a shoulder. c: C=O stretching in CCl4 occurs at a low wavenumber. d: As a result of hydrogen bonding with the hydroxy groups the νC=O band of the 3-keto groups appears at relatively low frequency, and thus theνC=O band cannot be observed.

25 Ethinylestradiol (炔雌醇) IR spectrum
υC=C1615，1590，1505cm－1苯环的骨架振动； υ≡CH 3300cm－1 炔的特征峰； υ－OH 3610cm－1游离酚羟基的伸缩振动； υ－OH 3505cm－1 C17- 羟基的伸缩振动。

26 IR spectrum of Dexamethasone(地塞米松)

27 Infrared spectrophotometry
Principles : Electromagnetic radiation ranging between 500 cm-1 and 4000 cm-1(2500 and nm)is passed through a sample and is absorbed by the bonds of the molecules in the sample causing them to stretch or bend. The wavelength of the radiation absorbed is characteristic of the bond absorbing it.

28 Infrared Radiation Unit Conversion
Wavelength Range :0.7~1,000µm(15,000~10cm-1) Energy Increase Infrared 1,000 μm 0.4 0.2 0.7 25 2.5 25,000 cm-1 50,000 cm-1 15,000 cm-1 10 cm-1 400 cm-1 4,000 cm-1 X-Ray UV VIS Microwave NIR Far IR Mid IR Wavelength Wave number Unit The unit of wave number(cm-1) is used for IR Spectroscopy Conversion Wave number (cm-1)=10,000 ÷ Wavelength (µm) Wavelength (µm)=10,000 ÷Wave number (cm-1)

29 Factors determining intensity in IR spectra:
The intensity with which a bond absorbs radiation depends on its dipole moment. The order of intensity of absorption is: C–O > C– Cl > C –N > C– C–OH > C–C–H 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=C–C=O > C=C–C=C > C=C–C–

30 Instrumentation 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 interferometer.

31 Michelson interferometer
Source Moving Mirror (M1) ０ λ/2 λ -λ/2 -λ Beam splitter (Half Mirror) To Sample compartment To Detector Interferometer Fixed Mirror(M2)

32 Michelson Interferometer
(Michelson: 1890’s) Fixed mirror X 50% Polychromatic Radiation source 50% 25% + 25% 50% 50% 100% 25%+ 25% Beamsplitter Moving mirror Detector

33

34 Applications 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 any other method. Can be used to characterize samples in the solid and semi-solid states such as creams and tablets.

35 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 that have different physical properties such as solubility and melting point which may be important in the manufacturing process).

36 Fields 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. Food Industry Forensic Science Polymers Oil Industry Materials Science IR Semiconductors IR Ceramic Pharmaceutical Industry Biological Science Environmental Science

37 Strengths Provides a complex fingerprint which is unique to the compound being examined. Computer control lf instruments means that matching of the spectrum of a compound to its standard fingerprint can now be readily carried out.

38 Limitations 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 degree 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.

39 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

40 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 products 、 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

41 举 例 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

42 Examples Dexamethasone sodium phosphate、hydrocortisone sodium phosphate——free phosphonic acid ——determination of A Dexamethasone sodium phosphate——methanol and acetone——GC Ethinyloestradiol ——estrone ——colorimetry Dexamethasone acetate、fluocinonide acetate——selenium——OCF-colorimetry Steroid hormones——other steroids——TLC、HPLC

43 Assays 四氮唑比色法（ Tetrazoline 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) Section 4

44 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。

45 Commonly used tetrazoline salts： Red tetrazoline(RT) :
Tetrazoline colorimetry Commonly used tetrazoline salts： Red tetrazoline(RT) : 2,3,5-triphenyltetrazolium chloride (TTC); Blue tetrazoline(BT): {3,3’-dianisole-bis[4,4’-(3,5-diphenyl) tetrazolium chloride]}

46 TTC BT

47 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。

48 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 振摇溶解 稀释至刻度 摇匀 对照品 溶液 100ml量瓶中 加无水乙醇 约30ml 精密称取 样品4g 水浴加热 搅拌溶解 冰浴冷却 滤过 烧杯中 加无水乙醇 摇匀 供试品 溶液 滤 液 稀释 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 供试品溶液 各精密量取1ml 对照品溶液 摇匀 放置40～45min 测A 计算 即得 25℃

49 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。

50 影响因素： 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。

51 Influence factors： A. Functional groups
They often influence the rate of reaction : C11-keto>C11-hydroxy；C21-hydroxy>C21-hydroxyl ester。 B.   Solvent and 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 tetrazoline salt first and then alkaline solution to prohibit the decomposition of corticosteroids.

52 Influence factors： 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 , faster the rate of color reaction is. Generally, room temperature or 30℃ constant temperature is the best condition. The result has a better repeatability.

53 Isoniazid colorimetric method
本法主要用于甾体激素制剂的测定。 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。 + + H2O

54 Kober Reaction 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 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.

55 Requirements 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。

56 铁酚试剂(Iron-phenol Reagent)
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。

57 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。

58 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 Δ4-3-keto corticoid hormones, androgens, progestogens and many per oral contraceptives have the maximum absorption at about 240nm.With the structure of benzoic ring , estrogens have the maximum absorption at about 280nm.

59 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。

60 HPLC Advantages: Rapid and efficient separation;
Small quantities of samples; High sensitivity Conditions: Reverse-phase: ODS silica gels; Mobile phase :methanol and water.In order to improve separation effect, acetic acid buffer or phosphate acid buffer is added to adjust the pH value of mobile phase. UV detector.

61 Brief Summary General structures:
Corticosterone , Androgen , Progestogen , Estrogen

62 Characteristics： Color reaction:
steroid ring — reaction with sulfuric acid C17-α- hydroxyacetone— reaction with tetrazoline salt Ketone — reaction with 2,4-dinitrophenyl hydrazine, isoniazide or phenylhydrazine sulfate Fluorine — reaction with alizarin complexone and cerous nitrato Phenol hydroxyl — reaction with diazobenzene sulfonic acid Precipitation reaction: C17-α- hydroxyacetone — reaction with Fihling reagent — reaction with silver nitrate preparing in ammonia Ethynyl — reaction with silver nitrate Organic combine chlorine — reaction with HNO3-AgNO3

63 Melting point determination of derivative
Hydrolysis product reaction Ultraviolet spectroscopy Conjugate systems (C=C－C=O,C=C－C＝C） Infrared spectroscopy

64 Specific impurity tests：
Dexamethasone sodium phosphate、hydrocotisone sodium phosphate— free phosphoric acid — determination of A Dexamethasone sodium phosphate—methanol and acetone—GC Ethinyloestradiol — estrone — colorimetry Dexamethasone acetate —selenium Steroid hormones——other steroids——TLC、HPLC Tetrazoline colorimetry Isoniazide colorimetric method Cober reaction colorimetry HPLC Assays:

65 Editor: Qiang Fu, Hongqin Ke
Thank you Editor: Qiang Fu, Hongqin Ke College of Pharmacy , Xi’an Jiaotong university