h第八章 生物碱类药物分析课件

1、第八章 生物碱类药物分析 Chapter 8 The Analysis of Alkaloids 药物分析课件系列Special identification testAssayAlkaloidsPropertyImpuritytest Classification contents fundamental requirements reviewContents The structures and properties of Alkaloids(6 groups);The primary identification tests of Alkaloids;Special impurity t

2、ests for the Alkaloids;Assays of Alkaloids.Fundamental requirements Mastering the theories, procedures and conditions: non-aqueous titration, extractive titration, and acid dye colorimetry; Understanding the structures and properties of Alkaloids;Understanding the specific impurity tests of Alkaloid

3、s;Understanding the applications of high performance liquid chromatographic and gas chromatographic methods in assays of Alkaloids. Familiarizing with the primary identification tests of Alkaloids;Fundamental requirements ephedrine hydrochloride pseudoephedrine hydrochloride colchicin The structural

4、 characteristic of this type alkaloids is that the N atom is in the alphatic chain rather than in aromatic ring. The typical drugs are Ephedrine hydrochloride, Pseudoephedrine hydrochloride, Colchicin. Benzoalkylamineatropine sulfate anisodamine hydrobromide Tropanes This type of alkaloids are the c

5、ondensation products of alkaloid derived from tropanes and different kinds of esters. The typical drugs are atropine and anisodamine.Isoquinolines morphine hydrochloride codeine phosphate The typical drugs are morphine, codeine, narcotine.Indoles reserpine strychnine nitrate The number of this type

6、of alkaloid is large. Many of them have complex structures and obvious physiological activity. Such as strychnine nitrate, reserpine etc. Property：Basicity: Generally, alkaloids show weak basicity. Nearly all of alkaloids can form salt with acids.Solubility: Most of alkaloids are lipophilic and thei

7、r salts can be dissolved in water.UV: Because of their highly conjugated system, most of the alkaloids have UV absorption.d. ChiralityGo to reviewXanthines caffeine theophylline Xanthines include many alkaloids, such as caffeine and theophylline. In structure, they have 4 N atoms, but all of them sh

8、ow weak basicity because of the influence from the ortho-position carbonyl group electron absorbing effect. They are difficult to form salt, So, in clinic, we use caffeine free base.Identification Tests General identification testBiuret reactionVitaili reactionThakkeioquin reactionMarquis reaction;

9、Frohde reaction; Murexide reactionMeasurement of melting pointColor reaction Precipitation reactionUltraviolet absorption spectroscopyInfrared spectrometryChromatographySpecific identification test Color reactionConcentrated sulfuric acid, concentrated nitric acid, molybdenum sulfuric acid, vanadium

10、 sulfuric acid, selenium sulfuric acid etc.are commonly used as the reaction agents. The mechanism may be involved in dehydration, oxidation, condensation etc.General identification testsPrecipitation reaction Alkaloids can form insoluble or undissolved salts, compounds or complex salts by react wit

11、h heavy metallic salt(bismuth potassium iodide, potassium mercuric iodide, etc.) or macro-molecule acid precipitating agents (phosphomolybdic acid, silicotungstic acid etc.) in acidic water solution.General identification testsUltraviolet absorption spectroscopy Most of alkaloids have aromatic ring

12、and conjugate double bond structure, so they have one or more characteristic peaks in ultraviolet band for identification. General identification tests Press here to see the commonly used methodsCommonly used methodsComparing with maximum absorbance wavelength, minimum absorbance wavelength and corr

13、esponding variance of absorbance. e.g Identification of colchicineChP(2000)秋水仙碱2. Comparing uniformity of sample absorption spectrum with that of standard. e.g Identification of emetineUSP(24)吐根碱3. Compare uniformity of absorbance index. e.g ReserpineJP(14)利血平. Infrared spectrometry Infrared absorpt

14、ion spectral method is also regarded as one of the primary identification method. The alkaloids identified by infrared absorption spectral method and noted in ChP(2000) are reserpine, morphine, atropine, colchicine, etc. General identification testsChromatography TLC is the major method for identifi

15、cation of alkaloids, and HPLC is also used. Presently, silica gel is the commonly used sorbent. For effective separation, alkaloids should be in their free base form. On the contrary, if the alkaloids in salt form, they would be held so tightly that the spots show serious tailing. The following meth

16、ods are commonly used to overcome the problem. General identification testsSpecific identification tests 双缩脲反应(Biuret reaction):芳环侧链具有氨基醇结构生物碱药物的特征反应。Vitaili reaction: 托烷类生物碱莨菪酸结构的特征反应，与发烟硝酸得黄色三硝基衍生物，遇醇制氢氧化钾即显深紫色。绿奎宁反应(Thakkeioquin reaction): 6位含氧喹啉衍生物的反应。可在其盐类的微酸性水溶液中，滴加溴水和氯水，至微过量时，再加入过量的氨水，应呈翠绿色反应

17、。Go to reviewMurexide reaction 黄嘌啉生物碱的特征反应，即样品加盐酸和氯酸钾，在水溶液上共热蒸干，此残渣遇氨气呈紫色，再加氢氧化钠溶液颜色即消失。Marquis reaction 含酚羟基异喹啉类生物碱的特征反应。该类药物遇甲醛硫酸可形成具有醌式结构的有色化合物。Frohde reaction 系吗啡生物碱的特征反应。 吗啡加钼酸试液即显紫色，继而蓝色，再为棕绿色。The Special Impurities in the Alkaloids Drugs Impurity atropine sulfate tropine, other alkaloids quin

18、ine sulfate undissolved in chloroform- ethanol，other chinines morphine hydrochloride apomorphine，narceine，other alkaloids codeine phosphate morphine strychnine nitrate strychnine reserpine oxidation product Commonly used examination methodsPhysical tests Difference in Color Difference in optical act

19、ivity Difference in spectrum Chemical reaction tests Color difference while react with some reagentsChromatography TLC HPLC Difference in absorptionSpecial Impurity TestsGo to reviewAssay Commonly used methods： Non-aqueous titration Extractive titration Acid dye colorimetry Ultraviolet spectroscopy

20、ChromatographyNon-aqueous titration： Theory Non-aqueous titration is the most common volumetric method used in assays. It is suitable for the titration of very weak acids or bases and provides a solvent in which organic compounds are soluble. The most commonly used procedure is the titration of orga

21、nic bases with perchloric acid in acetic acid. These assays sometimes take some perfecting in terms of being able to judge precisely the end-point. Theory Water behaves both as a weak acid and a weak base thus in an aqueous environment it can compete effectively with very weak acid and bases with re

22、gard to proton donation and acceptance as shown in Figure 1. The effect of this is that the inflection in the titration curves for very weak acids and very weak bases is small because they approach the pH limits in water of 14 and 0 respectively thus making end-point detection more difficult. A gene

23、ral rule is thatH2O + H+ H3O+ Competes with RNH2 + H+ RNH3+ H2O + B OH- + BH+ Competes with ROH + B RO- + BH+Fig.1Competition of water with weak acid and bases for protonbases with pKa7 cannot be determined accurately in aqueous solution.Various organic solvents may be used to replace water since th

24、ey compete less effectively with the analyte for proton donation or acceptance.Non-aqueous titration of weak basesAcetic acid is a very weak proton acceptor and thus does not compete effectively with weak bases for protons. Only very strong acids will protonate acetic acid appreciably according to t

25、he equation shown below: CH3COOH + HA CH3COOH2+ + A-Perchloric acid is the strong of the common acids in acetic acid solution and the titration medium usually used for non-aqueous titration of bases is perchloric acid in acetic acid.Addition of acetic anhydride, which hydrolyses to acetic acid, is u

26、sed to remove water fromaqueous perchloric acid. Weak bases compete very effectively with acetic acid for protons.Crystal violet (very weak bases) or quinalidine red is used as the indicator in this type of titration. A typical analysis is shown in Figure 2 for L-DOPA. CH3COOH2+ CH3COOH Fig.2Analysi

27、s of DOPA by non-aqueous When the bases is in the form of a salt of a weak acid, removal of an anionic counter ion prior to titration is not necessary, e.g. for salts of bases with weak acids such as tartrate, acetate or succinate. However, when a base is in the form of a chloride or bromide salt, t

28、he counter ion has to be removed prior to titration. This is achieved by addition of mercuric acetate; the liberated acetate is then titrated with acetous perchloric acid. This is illustrated in Figure for the example of phenylephrine.HCl.Non-aqueous titration with acetous perchloric acid is used in

29、 the pharmacopoeial assays of : adrenaline, codeine, chlorpromazine.HCl , amitriptyline. HCl, propranolol. HCl, and quaternary amine salts such as neostigmine bromide. Cl Hg(CH3COO)2 + 2Cl HgCl2 + 2CH2COO2CH3COOH2+ + 2CH3COO 4CH3COOH Fig.3The analysis of phenylephrine hydrochloride Non-aqueous titra

30、tion of weak acidsFor the non-aqueous titration of weak acids a solvent such as an alcohol or an aprotic, solvent is used that does not compete strongly with the weak acid for proton donation.Typical titrants are lithium methoxide in methanol or tetrabutyl ammonium hydroxide in dimethylformamide. En

31、d-point detection may be carried out with thymol blue as an indicator or potentiometrically. Non-aqueous titration of acidic groups is carried out in pharmacopoeial assays of: barbiturates, uracils and sulphonamides. Condition: Alkaloids usually have weak bacisity,and there is no satisfactory result

32、 when titrated with acid directly in aqueous because of unobvious rising. But in non-aqueous acid conditions, if Kb is above 10-10, the basicity could be raised to the level of solvent Ac- with glacial acetic acid, and therefore, the basicity strengthens obviously. This method is usually to determin

33、e the contents of alkaloids in raw materials. Theoretics of non-aqueous titrationNon-aqueous titrationPrinciple: The titration of alkaloids is a replacement process, in fact, that is, strong acid replaces the weak acid combined with alkaloids. BH+A- + HClO4 BH+ ClO4- + HABH+A- stands for salts of al

34、kaloids, HA stands for replaced weak acid. The different acidity of replaced HA makes the different influences to the titration, so the corresponding conditions should be adopted according to the different instance. Non-aqueous titrationGo to reviewMethod： Adding glacial acetic acid 1030ml to sample

35、 which is dried properly till dissolution. If it is salt of halogen acid, put 5 glacial acetic acid solution of mercuric acetate 35ml, titrate with 5% perchloric reagent to endpoint, and correct the result with blank test. Non-aqueous titrationDiscussion: The range of application：KbHBrH2SO4HClHSO4-H

36、NO3Method of indicating endpoint: Electric potential method and indicator method; crystal violet is commonly used as the indicator.Non-aqueous titrationApplication： 氢卤酸盐的测定： 由于氢卤酸在冰醋酸中酸性较强，对测定有干扰，必须先加入过量的醋酸汞冰醋酸溶液，使形成难以电离的卤化汞，而氢卤酸盐则转变为可测定的醋酸盐，然后用高卤酸滴定可得满意结果。 In order to overcome the disturbance of ha

37、logen acid, put excess mercuric acetate to form mercuric halide which is difficult to ionize in the solvent, then the alkaloid salt of halogen acid transforms to a measurable acetate and the acceptable result could be attained for titration by perhalogen acid.Non-aqueous titrationB. The assay of sul

38、fate： 硫酸为二元酸，在水溶液中能完成二级电离，生成SO42-,但在冰醋酸介质中，只能解离为HSO4-，不再发生二级解离。因此，生物碱的硫酸盐，在冰醋酸的介质中只能被滴定至生物碱的硫酸氢盐(BH+)2SO42- + HClO4 BH+ClO4- + BH+HSO4- 测定生物碱硫酸盐时，应注意分子中氮原子的碱性强弱，方能正确地进行结果计算；并能正确地理解各测定方法项下滴定度的由来。如：硫酸阿托品USP(24)。Non-aqueous titrationASSAY - Dissolve about 1 g of Atropine Sulfate, accurately weighed, in

39、 50 mL of glacial acetic acid, and titrate with 0.1 Mol/L perchloric acid VS, determining the endpoint potentiometrically. Perform a blank determination, and make any necessary correction. Each mL of 0.1 Mol/L perchloric acid is equivalent to 67.68 mg of (C17H23NO3)2H2SO4. The assay of nitrate: 硝酸在冰

40、醋酸中是弱酸，但它具有氧化性使指示剂变色，所以用非水溶液测定生物碱硝酸盐时，一般不采用指示剂而用电位法指示终点。 Nitric acid shows weak acidity in glacial acetic acid, but its oxygenation would colorize the indicator. So, we usually use potentiometric method rather than indicator when titrating alkaloids nitrate in non-aqueous solution. Non-aqueous titra

41、tion The assay of preparations: Injections： Take some sample into water bath and make it to dryness, then dry it in a certain temperature and treat it according to the item of raw material. Tablets： In most of tablets, the excipient such as stearate, carboxymethylcellulose sodium etc always consume

42、perchloric acid. So, the samples should be extracted, separated and dried before assay.Non-aqueous titrationExtractive titrationCondition: Some alkaloids with strong basicity (pKb69) can be determined directly by acid/base titration after basifying and extracting by organic solvent.Principle:Alkaloi

43、d salts are dissolved in water,while free alkaloids are dissolved in organic solvent. With these properties, alkaloids are extracted and titrated. samplewater or dil. mineral acid solutionalkaline reagentdissociative alkaloidsorganic solventextract alkaloids water alkaloids alkaline reagentwater sol

44、uble impuritywashanhydrous sodium sulfate dehydratepure alkaloidswaterMethod:direct titrationback titrationback titration with base after extracted with acidExtractive titrationGo to reviewDiscussion: Alkalinizing reagent Commonly used reagent：ammonia, sodium bicarbonate, sodium hydroxide, calcium h

45、ydroxide and magnesium oxide. Strong bases are not suitable for the dissociation of the following alkaloids: 1Drugs with the structure of ester. e.g. atropine and reserpine, as they are easily decomposed. 2Drugs with phenol structure. e.g. morphine, are easily to form salt with alkalinizing reagent

46、and dissolved in water. 3Drugs containing fatty excipients are easily to be emulsified and make the extraction difficult and incomplete.Extractive titrationB. Extraction solvent: Selection rules： 1 Alkaloids are easily dissolved in the solvent, and the solvent is undissolvable with water； 2 It is in

47、ert to alkaloids and basifiers； According to the above rules, except for few alkaloids such as morphine which is undissolved in chloroform, the most efficient and commonly used extraction solvent is chloroform. Ethyl ether is another commonly used extraction solvent. Discussion: Extractive titration

48、C. Indicator selection The basicity of alkaloids is not strong enough.If it is titrated with strong acid,the formed salt shows acidity. So the indicator selected should change color in acid range. D. Prevention of emulsificationchenshi:chenshi:Discussion: Extractive titrationPrevention of emulsifica

49、tion： 1. Using weak basifier; 2. Using proper extraction solvent which does not emulsify easily; 3. Avoiding shaking fiercely.Application： This method is used to determin morphine in opium. There are more than thirty kinds of alkaloids in opium. The most effective components is morphine, and its con

50、tent is 10%. Then in sequence, narcotine(about 29), codeine(about 0.32), papaverine(about 0.8).Extractive titrationMethod： 供试品滤 液醚层水层精密称定，加水研磨，加入Ca(OH)2，定溶过滤 除去可待因，罂粟碱吗啡，罂粟酸，硫酸等形成可溶性钙盐 加入乙醇，乙醚，氯化胺，振摇使分层那可汀等易溶于有机溶剂的阿片生物碱吗啡结晶醚液洗涤，中性甲醇重结晶，用硫酸滴定液溶解溶液硫酸吗啡溶于水中，过量硫酸黄色溶液甲基红试剂，NaOH滴定液滴定Extractive titrationAc

51、id-dye colorimetry:Conditions：This method is commonly used in small dosage samples (especially injections) or quantitative analysis of alkaloids in organism.Principle： In proper medium, alkaloids can combine with hydrogen ion to form positive ion (BH+ ). Some acid-dyes, such as bromophenol blue, bro

52、mcresol green and so on，can disassociate to be negative ion (In- ). The positive and negative ions can quantitatively combine to form ion pair, which can be quantitatively extracted by organic solvent and have a absorption at certain wavelength. We can calculate the content of alkaloid by determinin

53、g the sample absorbance. BH+ + In- (BH+In-)water phase (BH+In-)organic phase Acid dye colorimetry:BH+In-organic phase BH+water phaseIn-water phase E= BH+ water phase In- water phase (BH+In-)organic phase Discussion： The key points of acid dye colorimetry are the formation of ion-pair and extraction procedure. The extraction efficiency could be