h第八章-生物堿類藥物分析

1、第八章 生物堿類藥物分析 Chapter 8 The Analysis of Alkaloids,,,藥物分析課件系列,Special identification test,Assay,Alkaloids,Property,Impuritytest,Classification,contents,fundamental requirements,review,Contents,The structures and pro

2、perties of Alkaloids(6 groups);The primary identification tests of Alkaloids;Special impurity tests for the Alkaloids;Assays of Alkaloids.,Fundamental requirements,Mastering the theories, procedures and conditions:

3、 non-aqueous titration, extractive titration, and acid dye colorimetry; Understanding the structures and properties of Alkaloids;Understanding the specific impurity tests of Alkaloids;,Understanding the applications

4、of high performance liquid chromatographic and gas chromatographic methods in assays of Alkaloids. Familiarizing with the primary identification tests of Alkaloids;,Fundamental requirements,Benzoalkylamines Tr

5、opanesQuinolines IsoquinolinesIndoles Xanthines,Classification,Press here to see the detailed classification,Go to review,ephedrine hydrochloride,pseudoephedrine hydr

6、ochloride,colchicin,The structural 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 hydrochlor

7、ide, Colchicin.,Benzoalkylamine,atropine sulfate,anisodamine hydrobromide,Tropanes,This type of alkaloids are the condensation products of alkaloid derived from tropanes and different kinds of esters. The typical drugs

8、are atropine and anisodamine.,Quinolines,quinine sulfate,quinidine sulfate,The commonly used drugs are quinine sulfate, quinidine sulfate etc.,·,·,Isoquinolines,morphine hydrochloride,codeine phosphate,The typ

9、ical drugs are morphine, codeine, narcotine.,Indoles,reserpine,strychnine nitrate,The number of this type of alkaloid is large. Many of them have complex structures and obvious physiological activity. Such as strychnine

10、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 their salts can be dissolved in wa

11、ter.UV: Because of their highly conjugated system, most of the alkaloids have UV absorption.d. Chirality,Go to review,,生物堿,來源于氨基酸,來源于異戊烯,,,來源于萜類,來源于甾體,,來源于鳥氨酸,,,吡咯類吡咯里西丁類托品烷類哌啶類吲哚里西丁類喹諾里西丁類喹啉類吖啶酮類簡單苯丙胺類四氫異喹啉類

12、芐基四氫異喹啉類生物堿苯乙基四氫異喹啉類生物堿芐基苯乙胺類生物堿吐根堿類生物堿簡單吲哚堿類簡單 β–卡波林類半萜吲哚堿類單萜吲哚堿類單萜生物堿倍半萜生物堿二萜生物堿三萜生物堿孕甾烷生物堿環(huán)孕甾烷生物堿膽甾烷生物堿,來源于賴氨酸,,,,,,,,來源于色胺酸,來源于苯丙氨酸/酪氨酸,來源于鄰氨基苯甲酸,,,,,,,Xanthines,caffeine,theophylline,Xanthines include

13、 many alkaloids, such as caffeine and theophylline. In structure, they have 4 N atoms, but all of them show weak basicity because of the influence from the ortho-position carbonyl group electron absorbing effect. They ar

14、e difficult to form salt, So, in clinic, we use caffeine free base.,Identification Tests,General identification test,Biuret reactionVitaili reactionThakkeioquin reactionMarquis reaction; Frohde reaction

15、; Murexide reaction,Measurement of melting pointColor reaction Precipitation reactionUltraviolet absorption spectroscopyInfrared spectrometryChromatography,Specific identification test,General ident

16、ification testsMeasurement of melting point theophylline, codeine phosphate in ChP(2000) and theophylline in BP(2002) are identified by the measurement of melting point.,Color reactionConcentrated sulfuric acid,

17、concentrated nitric acid, molybdenum sulfuric acid, vanadium sulfuric acid, selenium sulfuric acid etc.are commonly used as the reaction agents. The mechanism may be involved in dehydration, oxidation, condensation etc.

18、,General identification tests,Precipitation reaction Alkaloids can form insoluble or undissolved salts, compounds or complex salts by react with heavy metallic salt(bismuth potassium iodide, potassium mercuric iodid

19、e, etc.) or macro-molecule acid precipitating agents (phosphomolybdic acid, silicotungstic acid etc.) in acidic water solution.,General identification tests,Ultraviolet absorption spectroscopy Most of alkaloids

20、 have aromatic ring 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 methods,Com

21、monly used methods,Comparing with maximum absorbance wavelength, minimum absorbance wavelength and corresponding variance of absorbance. e.g Identification of colchicine[ChP(2000)秋水仙堿]2. Comparing uniformity o

22、f sample absorption spectrum with that of standard. e.g Identification of emetine[USP(24)吐根堿]3. Compare uniformity of absorbance index. e.g Reserpine[JP(14)利血平].,Infrared spectrometry Infrared absorp

23、tion 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

24、.,General identification tests,Chromatography TLC is the major method for identification of alkaloids, and HPLC is also used. Presently, silica gel is the commonly used sorbent. For effective separation, a

25、lkaloids 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 methods are commonly used to overco

26、me the problem.,General identification tests,1. Adding a little base into developing solvent, such as ammonia, diethylamine, etc.2. Basifying the silica gel plate with base. Usually, by mix the sorbent with some sodi

27、um hydroxide or PBS in the plate preparation procedure.,method:,Specific identification tests 雙縮脲反應(yīng)(Biuret reaction):芳環(huán)側(cè)鏈具有氨基醇結(jié)構(gòu)生物堿藥物的特征反應(yīng)。Vitaili reaction: 托烷類生物堿莨菪酸結(jié)構(gòu)的特征反應(yīng)，與發(fā)煙硝酸得黃色三硝基衍生物，遇醇制氫氧化鉀即顯深紫色。綠奎寧反應(yīng)(Tha

28、kkeioquin reaction): 6位含氧喹啉衍生物的反應(yīng)?？稍谄潲}類的微酸性水溶液中，滴加溴水和氯水，至微過量時，再加入過量的氨水，應(yīng)呈翠綠色反應(yīng)。,Go to review,Murexide reaction 黃嘌啉生物堿的特征反應(yīng)，即樣品加鹽酸和氯酸鉀，在水溶液上共熱蒸干，此殘渣遇氨氣呈紫色，再加氫氧化鈉溶液顏色即消失。Marquis reaction 含酚羥基異喹啉類生物堿的特征反應(yīng)。該類藥物遇甲醛硫

29、酸可形成具有醌式結(jié)構(gòu)的有色化合物。Frohde reaction 系嗎啡生物堿的特征反應(yīng)。 嗎啡加鉬酸試液即顯紫色，繼而藍色，再為棕綠色。,Special Impurity Tests,Most of the alkaloids are gotten from herbs, though some of them are synthesized. For the complex structure and long produ

30、ction process, the other alkaloids may present in the final products. On the other hand, alkaloids may be decomposed during storage. Because of the toxicity and activity of alkaloids, the impurities must be controlled st

31、rictly for safety.,The Special Impurities in the Alkaloids,Commonly used examination methodsPhysical tests Difference in Color Difference in optical activity Difference in spectrum Chemical reaction tests Co

32、lor difference while react with some reagentsChromatography TLC HPLC Difference in absorption,Special Impurity Tests,Go to review,Assay,Commonly used methods： Non-aqueous titration Extractive titrati

33、on Acid dye colorimetry Ultraviolet spectroscopy Chromatography,Non-aqueous titration：,Theory Non-aqueous titration is the most common volumetric method used in assays. It is suitable for the titration o

34、f very weak acids or bases and provides a solvent in which organic compounds are soluble. The most commonly used procedure is the titration of organic bases with perchloric acid in acetic acid. These assays sometimes tak

35、e 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

36、 with regard 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 limi

37、ts in water of 14 and 0 respectively thus making end-point detection more difficult. A general rule is that,H2O + H+ H3O+

38、 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 proton,,,,,bases with pKa7 cann

39、ot be determined accurately in aqueous solution.Various organic solvents may be used to replace water since they compete less effectively with the analyte for proton donation or acceptance.Non-aqueous titration of weak

40、 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 the equation shown below:

41、 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

42、acid.Addition of acetic anhydride, which hydrolyses to acetic acid, is used to remove water fromaqueous perchloric acid. Weak bases compete very effectively with acetic acid for protons.Crystal violet (very weak bases

43、) 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.2Analysis of DOPA by non-aqueous,When the bases is in the form of

44、 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 chlo

45、ride or bromide salt, the 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 Fi

46、gure for the example of phenylephrine.HCl.,Non-aqueous titration with acetous perchloric acid is used in the pharmacopoeial assays of : adrenaline, codeine, chlorpromazine.HCl , amitriptyline. HCl, propranolol. HCl, and

47、 quaternary amine salts such as neostigmine bromide.,＋ Cl－,Hg(CH3COO)2 + 2Cl－ HgCl2 + 2CH2COO－,2CH3COOH2+ + 2CH3COO－ 4CH3COOH,,Fig.3The analysis of phenylephrine hydrochloride,,,Non-aqueous titration of w

48、eak 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 methox

49、ide in methanol or tetrabutyl ammonium hydroxide in dimethylformamide. End-point detection may be carried out with thymol blue as an indicator or potentiometrically. Non-aqueous titration of acidic groups is carried ou

50、t in pharmacopoeial assays of: barbiturates, uracils and sulphonamides.,Condition: Alkaloids usually have weak bacisity,and there is no satisfactory result when titrated with acid directly in aqueous because of unobv

51、ious 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

52、usually to determine the contents of alkaloids in raw materials.,Theoretics of non-aqueous titration,Non-aqueous titration,Principle: The titration of alkaloids is a replacement process, in fact, that is, strong acid re

53、places the weak acid combined with alkaloids. BH+·A- + HClO4 BH+ ·ClO4- + HABH+·A- stands for salts of alkaloids, HA stands for replaced weak acid. The different acidity of replaced HA

54、 makes the different influences to the titration, so the corresponding conditions should be adopted according to the different instance.,,,Non-aqueous titration,Go to review,Method： Adding glacial acetic acid 10~30ml

55、to sample which is dried properly till dissolution. If it is salt of halogen acid, put 5％ glacial acetic acid solution of mercuric acetate 3~5ml, titrate with 5% perchloric reagent to endpoint, and correct the result wi

56、th blank test.,Non-aqueous titration,,Discussion:,The range of application：KbHBr>H2SO4>HCl>HSO4->HNO3Method of indicating endpoint: Electric potential method and indicator method; crystal violet is common

57、ly used as the indicator.,Non-aqueous titration,Application：,氫鹵酸鹽的測定： 由于氫鹵酸在冰醋酸中酸性較強，對測定有干擾，必須先加入過量的醋酸汞冰醋酸溶液，使形成難以電離的鹵化汞，而氫鹵酸鹽則轉(zhuǎn)變?yōu)榭蓽y定的醋酸鹽，然后用高鹵酸滴定可得滿意結(jié)果。 In order to overcome the disturbance of haloge

58、n 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 attain

59、ed for titration by perhalogen acid.,Non-aqueous titration,B. The assay of sulfate： 硫酸為二元酸，在水溶液中能完成二級電離，生成SO42-,但在冰醋酸介質(zhì)中，只能解離為HSO4-，不再發(fā)生二級解離。因此，生物堿的硫酸鹽，在冰醋酸的介質(zhì)中只能被滴定至生物堿的硫酸氫鹽,(BH+)2·SO42- + HClO4 BH+·Cl

60、O4- + BH+·HSO4-,測定生物堿硫酸鹽時，應(yīng)注意分子中氮原子的堿性強弱，方能正確地進行結(jié)果計算；并能正確地理解各測定方法項下滴定度的由來。如：硫酸阿托品[USP(24)]。,,Non-aqueous titration,ASSAY - Dissolve about 1 g of Atropine Sulfate, accurately weighed, in 50 mL of glacial acetic acid,

61、 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 m

62、g of (C17H23NO3)2·H2SO4.,,,The assay of nitrate: 硝酸在冰醋酸中是弱酸，但它具有氧化性使指示劑變色，所以用非水溶液測定生物堿硝酸鹽時，一般不采用指示劑而用電位法指示終點。 Nitric acid shows weak acidity in glacial acetic acid, but its oxygenation wou

63、ld colorize the indicator. So, we usually use potentiometric method rather than indicator when titrating alkaloids nitrate in non-aqueous solution.,Non-aqueous titration,The assay of preparations: Injections： Take

64、 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, carboxy

65、methylcellulose sodium etc always consume perchloric acid. So, the samples should be extracted, separated and dried before assay.,Non-aqueous titration,Extractive titration,Condition: Some alkaloids with strong basicity

66、 (pKb6~9) can be determined directly by acid/base titration after basifying and extracting by organic solvent.Principle:Alkaloid salts are dissolved in water,while free alkaloids are dissolved in organic solvent. With

67、these properties, alkaloids are extracted and titrated.,sample,water or dil. mineral acid,,solution,,alkaline reagent,dissociative alkaloids,,organic solvent,extract,alkaloids,,water,alkaloids,,,alkaline reagentwater so

68、luble impurity,wash,,anhydrous sodium sulfate,dehydrate,,pure alkaloids,water,Method:,,,direct titrationback titrationback titration with base after extracted with acid,Extractive titration,Go to review,Discussion:,Al

69、kalinizing reagent Commonly used reagent：ammonia, sodium bicarbonate, sodium hydroxide, calcium hydroxide and magnesium oxide. Strong bases are not suitable for the dissociation of the following alkaloids:

70、 [1]Drugs with the structure of ester. e.g. atropine and reserpine, as they are easily decomposed. [2]Drugs with phenol structure. e.g. morphine, are easily to form salt with alkalinizing reagent and dissolv