Karboksilne kisline

Analiza in nadzor zdravil

Fakulteta za farmacijo
Oktober 2009

1
 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
[ RCOO  ][ H 3O  ]
Ka = [ RCOOH ]

2
 Karboksilne kisline

O O H
O O

2,4-pentadion- enolna oblika-

ketonska oblika vinilogna karboksilna kislina

3
 Karboksilne kisline

O H
O H
H H H O
H H H
H H H
O H
H
H

4
 Karboksilne kisline

CH3 O
CH3 O

H3C
O H3C OH

CH3
O CH3
H

CH3 O

H O
H3C 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. 7
C. Infrared absorption spectrophotometry (2.2.24).
 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 water-bath 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.

11
 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]-2,5-diethyl-3,4-
dihydro-2H-pyran-6-yl]-2,3-dichlorophenoxy]acetic acid.

12
 Etakrinska kislina

Me Me
CH2
Cl HX Cl X

Cl Cl
O O

HO HO
O O
O O

X = -NH2
-NHR
-SR

13
 Me Me
CH2 OH
N
H2NOH H

O O

HO HO
O Cl O Cl
O Cl O Cl

O
H
O Et
H

Et HO H OH
O Cl N
- HCl
RO N O Cl
Cl OH

H+
-H2O

O O
H Et
Et
RO N RO N
H 14
Cl Cl
 Me
R1 CH2 OH R1 - CH2 OH
CH2 Cl Cl
Cl
-OH Cl Cl
Cl O O
O
HO R R
O
2a 2b
O
1
R R

Cl Cl

Cl Cl
H
- R1
1 + 2b O O R1
R1 R1
Cl Cl
-
OH CH2 O
Cl Cl
O O

R R

3a 3b

Cl
R Cl
R1 R1
H
O
H2O

- OH- + HCHO
O Cl

Cl
R

15
4
 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.

16
 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)

17
 Reakcija na laktate

OH O
O Br2
O O
+ CO2
OH OH H

H2C O
(CN)5Fe......N=O +
H

4-

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

18
Mlečna kislina

19
 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).

20
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.

21
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.

22
 Mlečna kislina; Lactic acid

OH
O O O

OH
O O

H O
O
n
OH O H OH
23
 Estri alifatskih in aromatskih karboksilnih kislin z
bazičnim centrom

H3C H3C H3C

COOH
N N N
H
H OH OH

OH H H

tropin psevdotropin ekgonin

24
 Estri alifatskih in aromatskih karboksilnih kislin z
bazičnim centrom
HO H
H
N H
N O
OH O2N CHO
=
H N
- H2O

Me
Me O H
H
HO N
O N
N O
O
OH
H
Me
H

25
 Epimerizacija

H Me
+ +
N N
Me OH H OH

H H

26
 Atropin, Atropine sulphate, Atropini sulfas
Me
N
H

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-2-phenylpropanoate]
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 2-phenylpropenoate
(apoatropine),

32
 Impurities

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

33
 Odcep vode iz atropina-nastanek apoatropina

Me Me
N N
H H
H+
O O
- H2O

O OH O CH2

34
 Vitali-Morinova reakcija za dokaz atropina
NO2

HNO3
R O H - HNO3
R O H
- HNO3
O OH
O O
1 NO2
2

NO2 NO2

+ H2O +OH-, - H2O

R O R O H
- H2O -OH-, + H2O
O CH2 O OH

3 4

O O O
+ + +
N O N O N O

R O R O R O
- -
O OH O OH O- OH

35
5a 5b 5c
 AMIDI IN IMIDI ALIFATSKIH KARBOKSILNIH
KISLIN-ETOSUKSIMID, ETHOSUXIMIDUM, 3-etil-3-
metil-2,5-pirolidinedion

H
O N O
OH- NH2
Me NH2
O COO- + -OOC O
Me Et
Me Me
Et

OH-

O
O COO-

Et
Me 36
 Etosuksimid-identifikacija

H HO OH
N O O O
O O
H2SO4 2
Me Me

Me Me - 2H2O

HO O OH O O O

OH-
O
- H2O Et Me
Me
Et O
O O

37
 O O H H R

+ H2O O H2 N COOH

O
O
- H2O
O H
O
9 10
H
O O O O
O
OH H
R N
N - CO2 N C R
H H R
O H - H2O
O OH

11 12a 12b

H2O

O
H
R
O
+ NH2 AMINOKISLINE-ninhidrinska
OH reakcija
13

O O
OH
13 + H2O OH
- NH3 H
OH 14 O

13 NH

15
O
10
14 + H2O

O O

15 +14 N
- H2O 38
O HO 16
 Aminokisline-ninhidrinska reakcija

O O O O

N N

O O O O

17

O O

N
-
O O

39
Levodopa, Levodopum, (2S)-2-amino-3-(3,4-
dihydroxyphenyl)propionic acid

HO
H
COOH

HO
NH2

40
 Levodopa
COCl O
H H
HO COO- HO H
Py O
+
NH3+ -HCl HN
HO HO NO2
NO2 O

O O
H H
HO HO
O O
HN - H2O N
HO HO
HO NO2 NO2

O O
HO - HO
Na2CO3
O O
N - N
O O O O
+
N N
O O
41
AROMATSKE KARBOKSILNE KISLINE-salicilna kislina

HO O HO O
H
H
O O

HO O HO O
Fe/3 Fe/3
-
O O

42
 Vinilogne karboksilne kisline-Askorbinska kislina,
Acidum ascorbicum
CH2OH
H OH
O
O

H
OH OH

OH

OH
R R
O O O O

polester ogljikove kisline vinilogni polester ogljikove kisline 43

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

CH2OH CH2OH
CH2OH
H H
O H
O - H, -e O O
O - H, -e O
.-
H +H, +e H
H +H, +e
OH OH O O
O
O

44
Vinilogni estri karboksilnih kislin
Grizeofulvin, Griseofulvinum (zdravilo proti patogenim
Cl
glivicam) Cl
Me MeO O
HO
MeO O H+
H+
O
O

OMe O Me
OMe O OMe

1 2

Cl
Cl HO +
HO MeO O
MeO O
OH
OH
+
OMe O Me
OMe O Me

4a 4b

H
O O OH O O OH
HO oksidacija O
O O
MeO O MeO O
Me Cl Me
Cl

5 6 45
 DERIVATI OGLJIKOVE KISLINE
URETANI IN TIOURETANI
O O
-
RO + -OH RO OH O N O N
NH2 - H2O
NH2
- ROH

Co2+

[Co(OCN)4]2-

46
 SEČNINE IN DERIVATI

+ H H
H O
O O
+ +
NH2 H2N NH2 H2N NH2
H2N

47
 SEČNINE IN DERIVATI

O
HN O + NH3
H2N NH2
izocianatna kislina

O O
HN O +
H2N NH2 H2N NH

O NH2

Biuret 48
AMINOGLIKOZIDI

49
 FENILALKILAMINI

+
NH2 NH3

HO O

H
O H H
OH O N
H H + Me
HO N
Me
O
-
HO
50
 H H

O O O
O O H+, H2O

- 2EtOH

OH HO
+ RNH2

-H2O
n

R
N

O N

H+
HO
+
NH
R
+
N

- H3O+

+
N N
R R
+
N N

51
 EFEDRIN

OH
NHMe
Me
H
H

52
 Ksantinski derivati
Kofein (Caffeine, Coffeinum)

O OH
6 H H
N N
HN N

O N N N
H HO N
9

Dobra vodotopnost purinskih derivatov in slaba

Vodotopnost ksantinskih derivatov-vzrok?
Protoniranje? Bazičnost-kislost?
53
 Ksantinski derivati

O R2
R1 N H
N N
N

O 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 OH- N

N O N N
O N
O

O
O O
N
N N
N
+
N N
N N
O

O
N

N+
N

N+ N

N
55
 Fenilbutazon
H
N H+
N
H

+ + OH
N 2X- +
N 2
n

N N
N N

O 56
 Piridoksin hidroklorid
CH2OH
O CH2OH

NH+

CH2OH CH2OH

HO CH2OH O CH2OH

NH+ N

CH2OH
HO CH2OH

UV spektri vodnih raztopin so odvisni od pH vrednosti

57