Karboksilne Kisline_2008

Karboksilne kisline Analiza in nadzor zdravil Fakulteta za farmacijo Oktober 2009

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Karboksilne kisline Kislost organske spojine H-A določajo: • elektronegativnost skupine A • faktorji, ki stabilizirajo po disociaciji nastali anion A• Položaj ravnotežja je funkcija razlike proste energije med posameznimi species, to je odvisen je od njihove relativne stabilnosti, npr:

∆ G = - RTlnKa = -2.3RTlogKa = 2.3RTpKa Ka =

[ RCOO −][ H 3O +] [ RCOOH ] 2

Karboksilne kisline

O

O

2,4-pentadionketonska oblika

O

H

O

enolna oblikavinilogna karboksilna kislina

3

Karboksilne kisline

H

H

O

O

H H

H

H

H

H O

H

H O

H H

H

H

4

Karboksilne kisline

CH3 H3C

O

CH3 O H3C

O

OH CH3

CH3

O H CH3 O H3C

H O

O 5

Etakrinska kislina

6

ETACRYNIC ACID Acidum etacrynicum C13H12Cl2O4 Mr 303.1 [58-54-8] DEFINITION [2,3-Dichloro-4-(2-methylenebutanoyl)phenoxy]acetic acid Content: 98.0 per cent to 102.0 per cent (dried substance). CHARACTERS Appearance: white or almost white, crystalline powder. Solubility: very slightly soluble in water, freely soluble in ethanol (96 per cent). It dissolves in ammonia and in dilute solutions of alkali hydroxides and carbonates. IDENTIFICATION First identification: C. Second identification: A, B, D, E. A. Melting point (2.2.14): 121 °C to 124 °C. B. Ultraviolet and visible absorption spectrophotometry (2.2.25). Solvent mixture: 103 g/l solution of hydrochloric acid R , methanol R (1:99 V/V). Test solution: Dissolve 50.0 mg in the solvent mixture and dilute to 100.0 ml with the solvent mixture. Dilute 10.0 ml of this solution to 100.0 ml with the solvent mixture. Spectral range: 230-350 nm. Absorption maximum: at 270 nm. Shoulder: at about 285 nm. Specific absorbance at the absorption maximum: 110 to 120. C. Infrared absorption spectrophotometry (2.2.24).

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ETACRYNIC ACID Acidum etacrynicum Comparison: etacrynic acid CRS . D. Dissolve about 30 mg in 2 ml of aldehyde-free alcohol R. Dissolve 70 mg of hydroxylamine hydrochloride R in 0.1 ml of water R, add 7 ml of alcoholic potassium hydroxide solution R and dilute to 10 ml with aldehyde-free alcohol R. Allow to stand and add 1 ml of the supernatant liquid to the solution of the substance to be examined. Heat the mixture on a waterbath for 3 min. After cooling, add 3 ml of water R and 0.15 ml of hydrochloric acid R . Examined in ultraviolet light at 254 nm, the mixture shows an intense blue fluorescence. E. Dissolve about 25 mg in 2 ml of a 42 g/l solution of sodium hydroxide R and heat in a water-bath for 5 min. Cool and add 0.25 ml of a mixture of equal volumes of sulphuric acid R and water R. Add 0.5 ml of a 100 g/l solution of chromotropic acid, sodium salt R and, carefully, 2 ml of sulphuric acid R . An intense violet colour is produced. 8

ETACRYNIC ACID Acidum etacrynicum TESTS ►Related substances. Liquid chromatography (2.2.29).◄ ►Solvent mixture: acetonitrile R, water R (40:60 V/V). Test solution. Dissolve 25 mg of the substance to be examined in the solvent mixture and dilute to 25.0 ml with the solvent mixture. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with the solvent mixture. Dilute 1.0 ml of this solution to 10.0 ml with the solvent mixture. Reference solution (b). Dissolve 5 mg of etacrynic acid for system suitability CRS (containing impurities A, B and C) in 5.0 ml of the solvent mixture. Column: —  size: l = 0.25 m, Ø = 4.0 mm; —  stationary phase: end-capped octadecylsilyl silica gel for chromatography R (5 µm); 9

ETACRYNIC ACID Acidum etacrynicum Limits: —  correction factors: for the calculation of contents, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.6; impurity B = 0.6; impurity C = 1.3; —  impurity C: not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); —  impurities A, B: for each impurity, not more than 1.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.15 per cent); —  unspecified impurities: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); —  total: not more than 8 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.8 per cent); —  disregard limit: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).◄

10

ETACRYNIC ACID Acidum etacrynicum ASSAY Dissolve 0.250 g in 100 ml of methanol R and add 5 ml of water R. Titrate with 0.1 M sodium hydroxide, determining the end-point potentiometrically (2.2.20). 1 ml of 0.1 M sodium hydroxide is equivalent to 30.31 mg of C13H12Cl2O4.

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IMPURITIES

Specified impurities: A, B, C. A. R = H: (4-butanoyl-2,3-dichlorophenoxy)acetic acid, B. R = CH2Cl: [2,3-dichloro-4-[2(chloromethyl)butanoyl]phenoxy]acetic acid, C. [4-[2-[4-(carboxymethoxy)-2,3-dichlorobenzoyl]-2,5diethyl-3,4-dihydro-2H-pyran-6-yl]-2,3-dichlorophenoxy]acetic acid.

12

Etakrinska kislina Me Cl Cl HO

O O

Me CH2

X

Cl

HX Cl

O HO

O

O O X = -NH2 -NHR -SR

13

Me

Me CH2

N H

H2NOH O HO

O O

O HO

Cl Cl

O O

Cl Cl

O

O

H Et

RO

N Cl

OH

HO

- HCl

Cl

O

H

Et H OH N

Cl

O

OH H+ -H2O O

O H

Et

Et RO

N Cl

RO Cl

N H

14

Me Cl Cl

Cl

CH2

R1

Cl

-OH

CH 2OH

R1

CH2OH

O

O R

O 2a

O

-

Cl

O

R

HO

Cl

2b

1

R

R

Cl

Cl

-

1 + 2b

O

Cl

Cl

R1

H R1

O

R1

R1 Cl

Cl OH

Cl

CH 2O

Cl

-

O

O R

R

3b

3a

Cl R

Cl

R1 R1

H O

H2O - OH-

O

+ HCHO

Cl

Cl R

4

15

2.3.1. IDENTIFICATION REACTIONS OF IONS AND FUNCTIONAL GROUPS ESTERS To about 30 mg of the substance to be examined or the prescribed quantity add 0.5 ml of a 70 g/l solution of hydroxylamine hydrochloride R in methanol R and 0.5 ml of a 100 g/l solution of potassium hydroxide R in ethanol (96 per cent) R. Heat to boiling, cool, acidify with dilute hydrochloric acid R and add 0.2 ml of ferric chloride solution R1 diluted ten times. A bluish-red or red colour is produced.

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LACTATES

Dissolve a quantity of the substance to be examined equivalent to about 5 mg of lactic acid in 5 ml of water R or use 5 ml of the prescribed solution. Add 1 ml of bromine water R and 0.5 ml of dilute sulphuric acid R . Heat on a water-bath until the colour is discharged, stirring occasionally with a glass rod. Add 4 g of ammonium sulphate R and mix. Add dropwise and without mixing 0.2 ml of a 100 g/l solution of sodium nitroprusside R in dilute sulphuric acid R . Still without mixing add 1 ml of concentrated ammonia R. Allow to stand for 30 min. A dark green ring appears at the junction of the two liquids. sodium nitroprusside=dinatrijevpentacianonotrozilferat(II)

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Reakcija na laktate OH

O O OH

Br2

O

O

OH

+ CO2

H

H2C

(CN)5Fe......N=O

O

+ H

4(CN)5Fe....... N

O O

n

18

Mlečna kislina

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LACTIC ACID Acidum lacticum DEFINITION Mixture of 2-hydroxypropanoic acid, its condensation products, such as lactoyl-lactic acid and polylactic acids, and water. The equilibrium between lactic acid and polylactic acids depends on the concentration and temperature. It is usually the racemate ((RS)-lactic acid). Content: 88.0 per cent m/m to 92.0 per cent m/m of C3H6O3. CHARACTERS Appearance: colourless or slightly yellow, syrupy liquid. Solubility: miscible with water and with ethanol (96 per cent). IDENTIFICATION A. Dissolve 1 g in 10 ml of water R. The solution is strongly acidic (2.2.4). B. Relative density (2.2.5): 1.20 to 1.21. C. It gives the reaction of lactates (2.3.1).

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TESTS Solution S. Dissolve 5.0 g in 42 ml of 1 M sodium hydroxide and dilute to 50 ml with distilled water R. Appearance. The substance to be examined is not more intensely coloured than reference solution Y6 (2.2.2, Method II). Ether-insoluble substances. Dissolve 1.0 g in 25 ml of ether R. The solution is not more opalescent than the solvent used for the test. Sugars and other reducing substances. To 1 ml of solution S add 1 ml of 1 M hydrochloric acid, heat to boiling, allow to cool and add 1.5 ml of 1 M sodium hydroxide and 2 ml of cupri-tartaric solution R. Heat to boiling. No red or greenish precipitate is formed. Methanol (2.4.24): maximum 50 ppm, if intended for use in the manufacture of parenteral dosage forms. Citric, oxalic and phosphoric acids. To 5 ml of solution S add dilute ammonia R1 until slightly alkaline (2.2.4). Add 1 ml of calcium chloride solution R. Heat on a water-bath for 5 min. Both before and after heating, any opalescence in the solution is not more intense than that in a mixture of 1 ml of water R and 5 ml of solution S.

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ASSAY Place 1.000 g in a ground-glass-stoppered flask and add 10 ml of water R and 20.0 ml of 1 M sodium hydroxide. Close the flask and allow to stand for 30 min. Using 0.5 ml of phenolphthalein solution R as indicator, titrate with 1 M hydrochloric acid until the pink colour is discharged. 1 ml of 1 M sodium hydroxide is equivalent to 90.1 mg of C3H6O3. LABELLING The label states, where applicable, that the substance is suitable for use in the manufacture of parenteral dosage forms.

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Mlečna kislina; Lactic acid OH O

O OH

O

H

O

O O

OH

O

O

n

H

OH

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Estri alifatskih in aromatskih karboksilnih kislin z bazičnim centrom H3C

H3C

H3C N

N

N

H

H OH

tropin

COOH

OH

OH H

H

psevdotropin

ekgonin

24

Estri alifatskih in aromatskih karboksilnih kislin z bazičnim centrom HO H N

H N OH

H O2N

=

H

N

- H2O Me

Me O

O

CHO

O H

HO N

N OH H

H N O H

O

Me

25

Epimerizacija Me

H

+

+

Me

N

OH H

H

N

OH H

26

Atropin, Atropine sulphate, Atropini sulfas

Me

N H O O

OH 27

Atropine sulphate Atropini sulfas

C34H48N2O10S,H2O Mr 695 [5908-99-6] DEFINITION Bis[(1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3-hydroxy-2phenylpropanoate] sulphate monohydrate. Content: 99.0 per cent to 101.0 per cent (anhydrous substance). CHARACTERS Appearance: white or almost white, crystalline powder or colourless crystals. Solubility: very soluble in water, freely soluble in ethanol (96 per cent). IDENTIFICATION

28

Atropine sulphate Atropini sulfas IDENTIFICATION

First identification: A, B, E. Second identification: C, D, E, F. A. Optical rotation (see Tests). B. Infrared absorption spectrophotometry (2.2.24). Comparison: atropine sulphate CRS . C. Dissolve about 50 mg in 5 ml of water R and add 5 ml of picric acid solution R. The precipitate, washed with water R and dried at 100-105 °C for 2 h, melts (2.2.14) at 174 °C to 179 °C. D. To about 1 mg add 0.2 ml of fuming nitric acid R and evaporate to dryness in a water-bath. Dissolve the residue in 2 ml of acetone R and add 0.1 ml of a 30 g/l solution of potassium hydroxide R in methanol R. A violet colour develops. E. It gives the reactions of sulphates (2.3.1). F.  It gives the reaction of alkaloids (2.3.1). 29

Atropine sulphate Atropini sulfas

TESTS pH (2.2.3): 4.5 to 6.2. Dissolve 0.6 g in carbon dioxide-free water R and dilute to 30 ml with the same solvent. Optical rotation (2.2.7): − 0.50° to + 0.05° (measured in a 2 dm tube). Dissolve 2.50 g in water R and dilute to 25.0 ml with the same solvent. Related substances. Liquid chromatography (2.2.29). Test solution. Dissolve 24 mg of the substance to be examined in mobile phase A and dilute to 100.0 ml with mobile phase A. Reference solution (a). Dilute 1.0 ml of the test solution to 100.0 ml with mobile phase A. Dilute 1.0 ml of this solution to 10.0 ml with mobile phase A. Reference solution (b). Dissolve 5 mg of atropine impurity B CRS in the test solution and dilute to 20 ml with the test solution. Dilute 5 ml of this solution to 25 ml with mobile phase A. Reference solution (c). Dissolve► the contents of a vial of atropine for peak identification CRS (containing impurities A, B, D, E, F, G and H) in 1 ml of mobile phase A.◄ Reference solution (d). Dissolve 5 mg of tropic acid R (impurity C) in mobile phase A and dilute to 10 ml with mobile phase A. Dilute 1 ml of the solution to 100 ml with mobile phase A. Dilute 1 ml of this solution to 10 ml with mobile phase A. Column:

30

System suitability: reference solution (b): —  resolution: minimum 2.5 between the peaks due to impurity B and atropine. Limits: —  correction factors: for the calculation of content, multiply the peak areas of the following impurities by the corresponding correction factor: impurity A = 0.6; impurity C = 0.6; —  impurities E, H: for each impurity, not more than 3 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.3 per cent); —  impurities A, B, C, D, F, G: for each impurity, not more than twice the area of the principal peak in the chromatogram obtained with reference solution (a) (0.2 per cent); —  unspecified impurities: for each impurity, not more than the area of the principal peak in the chromatogram obtained with reference solution (a) (0.10 per cent); —  total: not more than 5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.5 per cent); —  disregard limit: 0.5 times the area of the principal peak in the chromatogram obtained with reference solution (a) (0.05 per cent).

31

Impurities A. (1R,3r,5S)-8-methyl-8-azabicyclo[3.2.1]oct-3-yl 2phenylpropenoate (apoatropine),

32

Impurities B. (1R,3r,5S)-8-azabicyclo[3.2.1]oct-3-yl (2RS)-3hydroxy-2-phenylpropanoate (noratropine

33

Odcep vode iz atropina-nastanek apoatropina

Me

Me

N

N

H

H

H+

O O

O

- H2O OH

O

CH2

34

Vitali-Morinova reakcija za dokaz atropina NO2 HNO3

R O H O

OH

O

NO2

NO2 + H2O

+OH-, - H2O

R O H

-OH-, + H2O

- H2O O

O

CH2

OH 4

3

O

OH

+

R O

O

OH 5b

+

N O

N O

R O

-

5a

O

O

+

N O

O

NO2

2

R O

- HNO3

O

1

R O

- HNO3

R O H

-

O-

OH 5c

35

AMIDI IN IMIDI ALIFATSKIH KARBOKSILNIH KISLIN-ETOSUKSIMID, ETHOSUXIMIDUM, 3etil-3-metil-2,5-pirolidinedion

O

H N

O Me Me

OH-

NH2

NH2

O

COOMe

+

O

-OOC

Et

Me

Et

OH-

O O

COOMe

Et

36

Etosuksimid-identifikacija

H N

O

O Me

O

∆ H2SO4

O

Me

OH

O

2

Me

Me

HO

OH

HO

O

- 2H2O

O

O

O OH-

O Me Et

O

- H2O

Et O

Me O 37

O

O + H2O

O

H

H

O

H2N

R COOH

O

- H2O

H

O

O

10

9 O

H O OH N

O

H

O

O

H

R

H

O N

N C R H

- CO2 - H2O

R

O

11

OH

12a

12b H2O O

H R

+

AMINOKISLINEninhidrinska reakcija

NH2

O OH 13 O

O OH

13 + H2O

OH - NH3

H 14

OH

O

O NH

13

15

O 10 14 + H2O

O

O 15 +14

N

38

- H2O O

HO

16

Aminokisline-ninhidrinska reakcija O

O

O

N

N O

O

O

O

O

17

O

O N O

O

39

Levodopa, Levodopum, (2S)-2-amino-3(3,4-dihydroxyphenyl)propionic acid

HO H

HO

COOH

NH2

40

Levodopa H

HO

COCl COO+

HO

HO

Py -HCl

NH3+

H

O

HO HO

HO

O - H2O HO

NO2 O

O

HN

NO2

H

HO O

O NO2

O HO

N

O

N

HO

O Na2CO3

H

HN

HO

NO2

H

O

O O + N O

O

N

O O N O

41

AROMATSKE KARBOKSILNE KISLINE-salicilna kislina HO

O

HO

H

H

O

HO

O

Fe/3 O

O

HO

O -

O

Fe/3 O

42

Vinilogne karboksilne kislineAskorbinska kislina, Acidum ascorbicum CH2OH H

OH O

O

H OH OH

OH OH R

O

O

polester ogljikove kisline

R

O

O

vinilogni polester ogljikove kisline 43

Oksidacija askorbinske kisline do dehidroaskorbinske kisline je dvostopenjski proces, ki gre preko vmesnega radikala

CH2OH H

O H OH OH

CH2OH

CH2OH O

- H, -e +H, +e

H

H

O H O

O .-

O

O

- H, -e +H, +e

O

H O

O

44

Vinilogni estri karboksilnih kislin Grizeofulvin, Griseofulvinum (zdravilo proti Cl Cl HO patogenim glivicam) Me MeO O MeO

O

H+

O

O O Me

OMe

O OMe

OMe

2

1

Cl

Cl MeO

O

HO

MeO

O

OMe

O Me 4a

O

HO + OH

OH + OMe

O Me

4b

H

O

O OH

HO

oksidacija

O OH

O O

O O

MeO

Me

Cl 5

H+

O

MeO Cl

Me

6

45

DERIVATI OGLJIKOVE KISLINE URETANI IN TIOURETANI O

O + -OH

RO NH2

RO

OH NH2

- H2O - ROH

O

N

O

N

Co2+

[Co(OCN)4]2-

46

SEČNINE IN DERIVATI

H H2N

+ H O

O + NH2

H2N

NH2

O + H2N

H NH2

47

SEČNINE IN DERIVATI

O H2N

HN

NH2

O

+ NH3

izocianatna kislina

O HN

O

+

H2N

O NH2

NH

H2N O

Biuret

NH2 48

AMINOGLIKOZIDI

49

FENILALKILAMINI

+ NH3

NH2 HO

O

H O OH HO HO

H N

H

O

Me

H H N + Me

O 50

H O

O

O

O

H+, H2O

O

H

- 2EtOH

OH

+ RNH2

HO

-H2O

n

R N N

O H+

HO R

+

NH +

N - H3O+

R

N +

N

+

N

R N

51

EFEDRIN

OH NHMe Me H H

52

Ksantinski derivati Kofein (Caffeine, Coffeinum) O

OH 6

HN O

N H

H N N 9

H N

N HO

N

N

Dobra vodotopnost purinskih derivatov in slaba Vodotopnost ksantinskih derivatov-vzrok? Protoniranje? Bazičnost-kislost?

53

Ksantinski derivati O R1

O

R2 N

N N

N

H N

N N

N

R3 Purin (topnost v vodi 1 :2) kofein: R1=R2=R3=Me (topnost v vodi 1 : 60) Teofilin: R1=R3=Me, R2=H (topnost v vodi 1 : 120) Teobromin: R1=H, R2=R3=Me (topnost v vodi 1 : 3000) Ksantin: topnost v vodi 1: 15000

54

Kofein-dokazna reakcija O

O N

N N

O

N

N

OH-

N

N

O

N

O O

O

O

N

N

N

N + N

N

N

N

O

O

N N+

N N+

N

O N

55

Fenilbutazon H N

H+ N H

+ N

+ N

2X-

OH +

2

n

O

N O

N

N

N

56

Piridoksin hidroklorid CH2OH CH2OH

O

NH+

CH2OH

CH2OH

CH2OH

O

CH2OH

HO

N

NH+

CH2OH CH2OH

HO

N

UV spektri vodnih raztopin so odvisni od pH vrednosti 57