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AMODIAQUINE HYDROCHLORIDE

Iqbal Ahmad and Tauqir Ahmad

Department of Pharmaceutical Chemistry, Faculty of Pharmacy,


University of Karachi, Karachi-75270, Pakistan.

K. Usmanghani
Department of Pharmacognosy, Faculty of Pharmacy,
University of Karachi, Karachi-75270, Pakistan.

1.
2.
2.1
2.2
2.3
3.
4.
4.1
4.2
4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
5.
5.1
5.2
5.3

INTRODUCTION
DESCRIPTION
Name, Formula, Molecular Weight
Appearance, Color, Odor and Taste
Proprietary Names
SYNTHESIS
PHYSICAL PROPERTIES
Melting Point
Solubility
Completeness of Solution
Acidity
Water Content
Residue on Ignition
Chromatographic Purity
Ultraviolet Spectrum
Infrared Spectrum
Nuclear Magnetic Resonance Spectrum
Mass Spectrum
Complex Formation
QUALITATIVETESTS
Identification
Color Tests
Field Test

ANALYTICAL PROFILES OF DRUG SUBSTANCES


AND EXClPlENTS - VOLUME 21

43

Copyright G 1992 by Academic Press, Inc


All rights of reproduction reserved in any form

44

5.4
6.
6.1
6.2
6.3
6.4
7.
7.1
7.2
8.

IQBAL AHMAD, TAUQIR AHMAD. AND K . USMANGHANI

Impurity Test for 4-(7-Chloro-4-quinolylamino)phenol Hydrochloride


METHODS OF ANALYSIS
Titrimetric Analysis
Spectrophotometric Analysis
Fluorometric Analysis
Chromatographic Analysis
METABOLISM AND PHARMACOKINETICS
Metabolism
Pharmacokinetics

TOXICITY
ACKNOWLEDGEMENT
REFERENCES

1.

INTRODUCTION

Amodiaquine is a congener of chloroquine and is employed for the


treatment of overt malarial attacks and for suppression. Although it is
more active than chloroquine both in vitro and in vivo against certain
strains of Plasmodium fcipamm
with decreased sensitivity to
chloroquine, amodiaquine is not recommended for routine use in the
treatment of such infections (1). It appears that phenolic hydroxyl is
essential to the activity of amodiaquine since the removal of this group
depresses, and its methylation completely destroys antibacterial activity
(2). Amodiaquine has been synthesized and patented under the name of
Camoquin by Parke, Davis and Company in 1949 (3). It is used medicinally
in the form of its dihydrochloride.
2.

DESCRIPTION

2.1 Name, Formula, Molecular Weight

Amodiaquine hydrochloride is 4-(7-chloro-4-quinolylamino)-2(diethylaminomethyl) phenol dihydrochloride dihydrate (4).

AMODIAQUINE HYDROCHLORIDE

CaH2zCIN30,2HC1, 2H20

45

464.8

The CAS registry No. is 6398-98-7.


Official monographs for amodiaquine hydrochloride are given in
Argentinian (1966), British (1988), Brazilian (1977), Egyptian (1984),
French (1982), Indian (1985), International (1981) and United States
(1990) Pharmacopeias.
2.2 Appearance, Color, Odor and Taste
A yellow, odorless or almost odorless, crystalline powder with a bitter
taste (5).

2.3 Proprietary Names


CAMAQI, Camoquin, Flavoquine, Miaquin (6,7).

3.

SYNTHESIS

Burckhaiter et al. (8) synthesized amodiaquine (111) in 1948 by


condensing 4,7-dichloroquinoline (I) with 4-amino-2-diethylaminomethylphenol (11) in dilute hydrochloric acid (Figure 1). In a later method
(9), the alkylamino group was added as a last step.

,q+

IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

46

C1

H2N a c H 2 N ( q H d 2

OH

c1

II

10
dilute
0". 2 HCl
hours

,@2N(c2H5)2

111

OH

Figure 1. Synthesis of Amodiaquine

The free base was recrystallized from absolute ethanol and converted
into the dihydrochloride by treating with hot concentrated hydrochloric
acid.
4.

PHYSICAL PROPERTIES

4.1 Melting Point

It melts at about 158OC (7).


4.2 Solubility
It is soluble in 22 parts of water and in 70 arts of ethanol (96%),
practically insoluble in chloroform and ether &).

AMODIAQUINE HYDROCHLORIDE

4.3

47

Completeness of Solution
A solution of 200 mg in 10 ml of water is clear (10).

4.4 Acidity
The pH of a 2.0% w/v solution is 3.6 to 4.6 (4).
4.5 Water Content
Not less than 7.0% and not more than 9.0% (10)
4.6 Residue on Ignition
Not more than 0.2% (10)
4.7 Chromatographic Purity
Chromatographic purity of amodiaquine hydrochloride can be
examined on thin-layer plate coated with a 0.25 mm layer of silica gel G
using solvent system chloroform (saturated with ammonium hydroxide):
dehydrated alcohol (9: 1). Under short-wavelength ultraviolet light, the
chromatogram shows principal spot at about the same Rf value, and no
secondary spot, as obtained with the USP Amodiaquine Hydrochloride RS
(10).
4.8 Ultraviolet (UV) Spectrum
The ultraviolet spectra of amodiaquine and amodiaquine
hydrochloride have been reported by Sunshine (12) and Clarke (7)
respectively. The ultraviolet absorption characteristics are used for the
identification of these drugs (4,10,13). The absorption spectrum of
amodiaquine hydrochloride as a function of pH in the range 1-11.8 shows a
hypsochromic effect at 343 nm, a hyperchromic effect at 305 nm and the
isosbestic point at 323 rim (14). The effect of solvents and substitution on
the ultraviolet spectra of amodiaquine has been studied and the changes of
absorption bands E,K, and B discussed in detail (15).
The ultraviolet spectrum of amodiaquine hydrochloride in 0.1 M
hydrochloric acid was recorded on a Shimadzu 240 UV-Visible
spectrophotometer and is shown in Figure 2. The uv spectral data
reported for amodiaquine and amodiaquine hydrochloride are listed in
Table 1.

48

IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANGHANI

1.50

!r
1

Figure 2.

WAVELENGTH Cnml

Ultravlofet Spectrum of Amodluquinc Hydrochloride


InO.l M HCI

3 .0

AMODIAQUINE HYDKOCHLORIDE

49

Table 1

UV Spectral Data for Amodiaquine and Amodiaquine Hydrochloride


Compound

Solvent

Amodiaquine

0.1 M HCl

Amodiaquine
hydrochloride

Water

0.1 M HCI
0.1 M HCl

Aq. acid
Aq. alkali
0.1 M HCl

Amax, nm
283
237
247
224
342
343
223
237
343
237
343
273
287
223
237
342.5

A (l%,Icm)

Molar
Absorptivity

890
530
470

41370
24630
21850

394410
366

1831019060
17010

366
600

17010
27890

Ref.
12

4
11

7
7

836
489
369

38850
22710
17160

*Values determined by the authors.

4.9 Infrared (IR)Spectrum


The infrared spectrum of amodiaquine has been determined in KBr
disc (4). The principal peaks in the infrared spectrum of amodiaquine
hydrochloride (KBr disc) are reported at 1565, 815, 1535, 1255, 869, 847
cm" (7). Attenuated total reflectance infrared spectrum is used to detect
amodiaquine hydrochloride in the solid state as a layer of crystals on
adhesive tape. The method has been applied to the identification of the
drug in tablet formulations. Common excipients such as starch, and
lactose (absorption in the 1000 to 1200 cm-' region) do not interfere with
the method (16).
The infrared spectrum of amodiaquine hydrochloride as KBr disc was
obtained with a Shimadzu IR 460 Infrared spectrophotometer and is
shown in Figure 3. The assignments for characteristic bands are given in
Table 11.

5.a
..

4.0

0
L

7.0

60
I

8.g
I

5.0
I

10 0

zoo

15.0

:oo.o

0
0
0

20.0

00"

Figure 3. Infrared Spectrum of Amodiaquine Hydrochloride (KBr disc).

500

0.0

'I

Wave number (em-')

'1500

40 0

I 2000

60-0

3000

0
ID

000

80.0

51

AMODIAQUINE HYDROCHLORIDE

Table I1
IR Spectral Assignments for Amodiaquine Hydrochloride
Frequency, cm-1

3420
3 170
1615
1585,1540,1505
1448
1265,1207
1095
852,840

Assignment

- NH stretching
- OH stretching
C = C stretching (aromatic)
C =C, C = N stretching
in disubstituted quinoline
- CH2-N- deformation
C-OH stretching (aromatic)
C-Cl stretching (aromatic)
isolated CH deformation
in disubstituted quinoline

4.10 Nuclear Magnetic Resonance (NMR) Spectrum


The 'H-NMR and 13C-NMR spectra of amodiaquine hydrochloride in
DMSO-d6 were determined at 300 MHz and 75.4 MHz respectively on a
Bruker AM-300 NMR spectrometer using tetramethylsilane as reference
standard. The 'H-NMR determinations included spin decoupling
experiments, 2D J-resolved and COSY-45 measurements (Figure 4-6).
The 13C-NMR spectra comprised DEFT and hetero-nuclear (C-H
correlation) measurements (Figure 7-9). The spectral assignments are
listed in Table 111.

d,
.I'

11-1'

L
11

I0

..,. ,...9 0 ..

8 0

1 0

. . :.

,.

P O

.. ..
:

. . .. -*

. .

. ...

4
PTL

F l p r c I. Homonuclear Chemlul Shin Cormlaled tCoS)-Ul ~ I I - N S I R


Spectrum or Amodisqulnc 1i)dmchloridc

-,rPPM

e
w

L
I

. . ,. . .. - , ._..
I!

-3r .

Iv,

h ?,

BIO

..

, ,1, 1, , , , , , , (
7.0

1 ,

6,O

PAI

, , , , , , , , r*~ , , , , , , , , ( , , , , , ,
5 0

4.0

b u r r 6. Homonuclcar 211 J-Rcsolvcd N M R Spcclrum of Amodiaquinc It)drorhloridr..

, , , ,

I 0

2 0

--

LPr2

1 0

CH: CHdCH2

l,,c.3tii

14

1
C.ll..ll

C.5

I ICI.

cd

C.Y

C.2

I I

-I--

C4

11

A L

Figure R.

75 M l l r "c-NMR Off Resonance Decoupled Spectrum ofAmodiaquine Hydrofhloride

t'

-==i

7'1

-2

L.

'4
I

IQBAL AHMAD. TAUQIR AHMAD. AND K. USMANGHANI

58

Table I11

H- and 13C-NMR Chemical shifts and Coupling Constants for


Amodiaquine Hydrochloride
H-NMR
Chemical shift
(PP4

Proton

coupling
constant
(J in Hz)

C-NMR
Chemicalshift
(PPd
156.04
138.87

7.67 (1H,d)

25

7.35 (lH,dd)
4.22 (lH,d)
4.22 (lH,s)
3.09 (4H,q)
1.27 (6H,t)

5
6
7
910
1112

8.62.5
8.6

8.44 (lH,d)
6.82 (1Hd)

2
3

8.% (lH,d)
7.78 (lH,dd)

5
6

6.7
1.2
7.0
7.0

9.1
9.12.1

l30.00
115.60
128.30
116.85
49.11
46.19
8.41
142.91
100.41
117.54
126.19
127.04

138.15
8.17 (lH,d)

2.1

118.97

Carbon

1
2
3
4
5
6
7
910
1112
2
3
4
5
6
T
8

154.85

127.92

10

NH/OH

10.89

4.11 Mass Spectrum

The electron impact ionization spectrum of amodiaquine


hydrochloride obtained at 70 eV using a solid probe insertion is shown in
Figure 10. The spectrum was run on a Finnigan Mat 112s double focusing
mass spectrometer connected to a PDP 11/34 (DEC) computer system. It
shows a molecular ion peak M + at m/z 355. Since the molecule contains
one chlorine atom, M+ 2 peak appears at m/z 357. The proposed
fragmentation pattern and prominent ions are given in Table IV.

Figure 10. Electron Impact-Mass Spectrum of Amodiaquine Hydrochloride

IQBAL AHMAD. TAUQIR AHMAD. A N D K.USMANGHANI

60

Table 4
Proposed Fragmentation Pattern of Amodiaquine Hydrochloride
d z

Relative intensity %

355, 357

55.77, 16.83

283

43.05

Ion

NH

@CH2

OH

282

99.00

c'w
@CH
OH

I-

253

43.56

179

8.18

177

5.81

AMODIAQUINE HYDROCHLORIDE

61

4.12 Complex Formation

Amodiaquine hydrochloride forms 1:l and 1:2 complexes with ferrous


sulphate. The infrared spectra indicate that amodiaquine hydrochloride is
bonded to iron via N and 0 and that water molecules are coordinated to
iron (17). It forms a 1:2 complex with silver nitrate in alcoholic solutions.
The average stability constant, log K, for the complex is 7.7 and A E is
about 10.8 kcal/mol. (18).
The formation of 1:l ion association complex between amodiaquine
and Fast Green FCF or Orange I1 dye has been reported (19).
5.

QUALITATIVE TESTS

5.1

Identification (4)

5.1.1 Dissolve 0.1 g of amodiaquine hydrochloride in 10 ml of water and


add 2 ml of 2 M sodium hydroxide. Extract with two 20 ml quantities of
chloroform, wash the combined chloroform extracts with 5 ml of water, dry
with anhydrous sodium sulphate and evaporate to dryness. Dissolve the
residue in 2 ml of chloroform. The infrared absorption spectrum of the
resulting solution is concordant with the reference spectrum of
amodiaquine.
5.1.2 The light absorption in the range 240 to 360 nm of a 0.003% w/v
solution of amodiaquine hydrochloride in 0.1 M hydrochloric acid exhibits
a maximum only at 343 nm. The absorbance at 343 nm is about 1.1.
5.1.3 To 1 ml of a 2% w/v solution of amodiaquine hydrochloride add 0.5
ml of cobalt thiocyanate reagent. A green precipitate is produced.
5.1.4 Amodiaquine hydrochloride yields the reactions characteristic of
chlorides.
The identification tests of amodiaquine hydrochloride based on
comparison of infrared and ultraviolet absorption spectra, and reactions
of chloride are reported in USP (10).
5.2 Color Tests (7)

Amodiaquine hydrochloride gives a blue color with Folin-Ciocalteu


reagent. The Liebermanns test yields a black color. An orange color is

62

IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

produced when amodiaquine hydrochloride is treated with Millon's


reagent.

5.3 Field Test (20)


Amodiaquine base is extracted from urine into amyl acetate
immediately after alkalinization. The addition of bromophenol blue in 5%
boric acid to the organic phase causes a green to blue coloring, depending
on the concentration of the drug. The sensitivity of the test is 0.8 mg%.
5.4 Impurity Test for 4-(7-Chloro-4-quinolylamino)phenol Hydrochloride
(4)Carry out thin-layer chromatography, using silica gel G as the coating
substance, spread in a layer about 0.5 mm thick, and a solvent system
chloroform: butan-2-one: diethylamine (50:40: 10). Apply separately to the
chromatoplate 5 pl of each of two solutions in methanol containing (1)
10.0% w/v of the substance being examined and (2) 10.0% w/v of
amodiaquine hydrochloride BPCRS and 0.020% w/v of 4-(7-chloro4-quinolylamino) phenol hydrochloride BPCRS. After development
remove the plate, heat it at 105' for 10 minutes, spray with a freshly
prepared mixture of equal volumes of a 10% w/v solution of iron (111)
chloride and a 1% w/v solution of potassium hexacyanoferrate (111) and
examine immediately.
Any spot corresponding to 4-(7-chloro-4quinolylamino) phenol in the chromatogram obtained with solution (1) is
not more intense than the spot with lower Rfvalue in the chromatogram as
obtained with solution (2).
6.

METHODS OF ANALYSIS

6.1 Titrimetric Analysis


6.1.1 Nonaqueous titration
The BP method (4) for the assay of amodiaquine hydrochloride as pure
drug and in dosage forms is based on nonaqueous titration. A 0.2 g
quantity of amodiaquine hydrochloride is dissolved in a suitable volume of
anhydrous glacial acetic acid, 7 ml of mercury (11) acetate solution is
added and the solution titrated with 0.1 M perchloric acid to a green end
point using 1-naphtholbenzoin solution as indicator. In dosage forms, a

AMODIAQUINE HYDROCHLORIDE

63

quantity of the powdered material equivalent to about 0.2 g of


amodiaquine hydrochloride is dissolved in 30 ml of water and 5 ml of 2 M
sodium hydroxide is added. Amodiaquine base is extracted with three 30
ml quantities of chloroform, the combined chloroform extracts are washed
with 10 ml of water and evaporated to a volume of about 10 ml. To the
chloroform extracts, 40 ml of anhydrous glacial acetic acid is added and the
solution titrated with 0.1 M perchloric acid using 1-naphtholbenzoin
solution as indicator. Each ml of 0.1 M perchloric acid is equivalent to
0.02144 g of CaHzCIN30,2HCl.

Wu et d. (21) have described a simple, rapid and accurate method for

the nonaqueous titration of amodiaquine in dosage forms. A powdered


sample of 5 milliequivalent weight is dissolved in 7 ml of N hydrochloric
acid, made alkaline with 3 ml of 6 N sodium hydroxide, shaken with 30 ml
of chloroform for 10 minutes and with 1 g of tragacanth for another 2
minutes, filtered through adsorbent cotton, and titrated (20 ml) with 0.1 N
acetic perchloric acid to blue or green end point using crystal violet
solution as indicator. For pure chemicals, the digestion with acid and
alkali could be omitted. The results agree with those obtained by the
official method.
6.1.2 Titration with brominating agents

Amodiaquine can be determined in bulk and in dosage forms by a


titrimetric method based on reaction with 1,3-dibromo-5,5dimethylhydantoin or N-bromosuccinimide as the titrant. The mixture is
later treated with potassium iodide solution and the liberated iodine
titrated with sodium thiosulphate solution. The recovery is about 100%
(22).
A method for the determination of amodiaquine hydrochloride in
tablets by titration with N-bromosuccinimide has been developed (23).
The sample is dissolved in water, treated with an acetic acid solution of the
reagent and mixed with potassium iodide. The iodine released is titrated
with sodium thiosulphate solution. The relative standard deviation for the
titration is 2.12% and the recovery is 99.4 - 101.0%.
6.1.3 Titration with vanadium (V)

The determination of amodiaquine hydrochloride by oxidation with


ammonium metavanadate solution and back titration of the unconsumed
reagent with acidic iron (11) ammonium sulphate solution, using

64

IQBAL AHMAD, TAUQlR AHMAD. A V D K. USMANGHANI

N-phenylanthranilic acid as indicator has been reported (24). The


recovery of amodiaquine hydrochloride in the pure form and in
pharmaceutical preparations is 99.83% (standard deviation 0.49%) and
99.69% (standard deviation 0.78%) respectively. The method is of general
applicability and is quick and simple compared with the official methods.
6.2 Spectrophotometric Analysis
6.2.1 Ultraviolet spectrophotometry
The USP assay (10) of amodiaquine hydrochloride in pure form and in
tablets involves ultraviolet spectrophotometric determination. A quantity
of the drug equivalent to about 300 mg is dissolved in dilute hydrochloric
acid (1:lOO) to obtain a concentration of about 15 pg/ml. The absorbance
of this solution, along with a solution of undried USP Amodiaquine
Hydrochloride RS in the same medium having a known concentration of
about 15 pg/ml, is determined at 342 nm using dilute hydrochloric acid
(1:lOO) as the blank. The quantity, in mg, of CmH22CIN30, 2HC1 in the
portion of amodiaquine hydrochloride taken is calculated by the formula
20C (Ad&), in which C is the concentration, in pg/ml, calculated on the
anhydrous basis, of USP Amodiaquine Hydrochloride RS in the standard
solution and AU and As are the absorbances of the solution of
amodiaquine hydrochloride and the standard solution respectively. The
same method is applied to the assay of amodiaquine hydrochloride in
tablets after extraction of the base into chloroform and then re-extraction
with dilute hydrochloric acid (1:lOO).
Amodiaquine and primaquine can be quantitatively separated by
selective precipitation with 4 N ammonium hydroxide, followed by
determination of the two compounds at 342 and 282 nm respectively. The
method is valid upto primaquine - amodiaquine ratio of 1:40. Recoveries
of 98.30 - 100.11% have been reported (25). The presence of higher
amounts of amodiaquine yields low results in respect of primaquine as on
precipitation with ammonium hydroxide, the primaquine is trapped into
the precipitate of amodiaquine (26).
Hassan et al. (27) have developed a method for the simultaneous
determination of amodiaquine - primaquine mixtures in dosage forms.
The drugs are extracted with 0.1 N hydrochloric acid and absorbance of
the mixture is measured at 342 and 282 nm. The concentration of each
compound is calculated by solving two simultaneous equations. Excellent

AMODlAQUlNE HYDROCHLORIDE

65

recoveries from authentic samples are obtained and the method is suitable
for routine analysis.
6.2.2 Colorimetry
Amodiaquine hydrochloride is determined colorimetricallyby complex
formation, in aqueous solution, with bromophenol blue, bromocresol
green, bromocresol purple, and methyl orange, respectively. The complex
with bromophenol blue has the highest molar absorptivity. Recoveries are
more than 98.6% for all complexes, and the absorbance is linear with
concentration in the range 1-11 pglml. The absorption maxima for the
complexes occur at 420 nm except for the bromocresol purple complex
which exhibits maximum at 415 nm. The various complexes are extracted
with chloroform and absorbance is measured at the respective maxima for
quantitative determination (28).
A simple, sensitive, and selective method for the determination of
amodiaquine hydrochloride in tablets has been developed. It is based on a
color reaction with chloramine-T in the pH range 7.4- 8.0. The chromogen
is extracted with chloroform and the absorbance is measured at 442 nm.
Beers law is obeyed in the concentration range 1-200 p g / l . The
coefficient of variation has been found to be 0.64% and the recovery
ranges between 100.3 and 102.5%. Chloroquine phosphate or primaquine
phosphate do not interfere with the method (29).

Amodiaquine reacts with cobalt and thiocyanate to yield stable ternary


complexes. These complexes are readily extractable in nitrobenzene to
give a greenish-blue color with maximum absorption at 625 nm that can be
used for quantitative determination. The mean recoveries for authentic
samples of amodiaquine hydrochloride are 100.81 & 1.77% (p = 0.05).
Alternatively, determination of the cobalt content of nitrobenzene extract
by atomic absorption spectroscopy provides an indirect method for the
determination of the drug with a mean recovery of 99.99 2 2.16%. Both
the methods have been successfully applied to the assay of the drug in
pharmaceutical preparations (30).
A colorimetric method for the determination of amodiaquine in tablets
or powders has been reported (31). The drug is dissolved in 0.1 N
hydrochloric acid, treated with acidic ammonium reineckate, the
precipitate dissolved in acetone, and the absorbance measured at 525 nm.
The results compare favourably with those obtained by the official
methods.

66

IQBAL AHMAD. TAUQlK AHMAD. AND K. USMANGHANI

Amodiaquine hydrochloride has been determined in tablets by


dissolving it in water and treating with an acetic acid solution of
N-bromosuccinimide. An orange-yellow color is produced, whose
absorbance is measured at 450 nm. Beers law is obeyed in the
concentration range 15-160 pglml. The relative standard deviation for the
method is 1.44%, and the recovery is 99.7-100.9% (23).
Amodiaquine hydrochloride tablets have been assayed by a method
based on the reaction of the drug with 2,3-dichloro-S, 6-dicyanop-benzoquinone and measurement of the absorbance at 460 nm. The
color attains its maximum intensity after five minutes and remains stable
for at least one hour. Beers law is valid in the concentration range 1-4
mg/100 ml, and the recovery is 99.9-102.6% (32). Another colorimetric
method for the determination of amodiaquine in tablets depends on its
reaction with chloranilic acid in aqueous solution and measurement of the
absorbance at 522 nm. The absorbance is linear over the concentration
range 0.04 -0.20 mdml, and the recovery is 99.9-101.3% (33).
A simple, rapid and sensitive method for the colorimetric

determination of amodiaquine in bulk and in pharmaceutical preparations


has been reported by Sastry et al. (34). It is based on the reaction of
amodiaquine with potassium dichromate at pH 1.1 in the presence of
sulphanilamide, and measurement of the absorbance of resulting solution
at 510 nm. The color is stable for twenty-four hours. Beers law is obeyed
in the concentration range 20-120pg/ml. The relative standard deviation of
the method is 0.94%, and the recovery is 99.0-101.0%. Chloroquine
present even in ten-fold excess does not interfere with the determination.
A highly sensitive method is based on the complexation of
amodiaquine with ammonium molybdate. The bound molybdenum is
converted into its thiocyanate, reduced, and the absorbance of the colored
solution measured at 465 nm. The Beers law limits, molar absorptivity
and Sandells sensitivity for the amodiaquine complex are 50-300pg/25 ml,
1.75 x lo4 M 1cm-l and 0.026 &cm2 / 0.001 absorbance unit, respectively.
Recovery ranges from 98-101%. The color obtained is stable for
twenty-four hours and common excipients do not interfere with the
method (35).

Amodiaquine forms a colored ion association complex with Fast Green


FCF or Orange I1 dye. The stoichiometric ratio of the drug-dye complex
has been shown to be 1:l. The method can be applied to the assay of
amodiaquine in bulk and in pharmaceutical preparations. Sulphur

AMODIAQUINE HYDROCHLORIDE

67

containing drugs do not interfere with the determination (19).


6.3 Fluorometric Analysis
A fluorometric method for the determination of amodiaquine in
serum, plasma, or red cells has been reported (36). Amodiaquine is
extracted from alkalinized biological fluids, buffered, and heated to
produce a species with marked increase in fluorescence, which could be
measured. Standard curves prepared in serum and red cells are linear
between 50 and 3000 pgll. Reproducibility of the assay and recovery of
amodiaquine from serum and red cells is satisfactory. The specificity of
the assay and the nature of the induced fluorophor are not known.
6.4

Chromatographic Analysis

6.4.1 Thin-layer chromatography (TLC)

Amodiaquine can be separated and identified on silica gel G plates


using a number of solvent systems. The spots are visualized under
short-wavelength ultraviolet light or by spraying with acidified
iodoplatinate solution. The following Rr values (Table V) have been
reported (37).
Table V
Solvent Systems for TLC of Amodiaquine
Adsorbent

Silica gel G F w dipped


in 0.1 M KOH and dried

Solvent system

Methanol: ammonia
(1rn1.5)
Cyclohexane :toluene :
diethylamine
(751510)
Cbloroform:methanol
( 91)
Acetone

Rr

0.62
0.08

0.40

0.37

The application of principal components analysis to the TLC behaviour


of a large number of basic drugs including amodiaquine has been studied
(38). A two-component model explains 77% of the total variance in four

68

IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANCHANI

eluting mixtures. For the identification of unknowns, the method provides


a drastic reduction of the range of possibilities to a few drugs.
6.4.2 High-performance liquid chromatography (HPLC)
A variety of HPLC packing materials have been prepared and their
chromatographic properties evaluated for separating amodiaquine and
other basic drugs using a single mobile phase. The three most promising
packing materials are silica, a mercapto Pr modified silica and a Pr sulfonic
acid modification (39).
A simple and precise HPLC assay for quantitating amodiaquine in
tablets and biological fluids involves acid extraction of the drug from
tablets and chloroform extraction of its base from the biological fluids
after treatment with ammonia. A p-Bondapak Ph column is employed for
separation with a mobile phase comprising methanol : water: acetic acid
(25:25:1) (pH 2.3), using quinidine as the internal standard. The mean
recovery of the drug from tablets is 102.03%, while in the biological fluids,
it ranges from 85.2 to 104.6%. Interference from tablet excipients or
biological fluids is negligible (40).
A column liquid chromatographic method for the simultaneous
determination of chloroquine, amodiaquine and their monodesethyl
metabolites in human plasma, red blood cells, whole blood and urine has
been developed (41). The drugs and internal standards are extracted as
bases with dichloromethane and then re-extracted into an acidic aqueous
phase. Separation is achieved using a reversed-phase column and a mobile
phase of phosphate buffer (pH 3.0) : methyl cyanide (88:12). The
absorbance of the drugs is monitored at 340 nm with a sensitivity limit of
10 pmoVml. The mean overall recovery from each biological fluid is more
than 75%. This method can be applied to therapeutic, pharmacokinetic,
and epidemological studies.

7.

METABOLISM AND PHARMACOKINETICS

7.1 Metabolism
Churchill et al. (42) have isolated four metabolites of amodiaquine in
humans using a reversed-phase HPLC method. The two major metabolites
have been identified as desethylamodiaquine and 2- hydroxy-

AMODIAQUINE HYDROCHLORIDE

69

desethylamodiaquine. The importance of these metabolites in the


antimalarial effect of amodiquine in humans and on the in vitro sensitivity
of persons dosed with amodiaquine is discussed.
2-Hydroxydesethylamodiaquine has been isolated from urine and characterised by
HPLC and NMR spectroscopy. The presence of three additional
metabolites of this drug in humans has been suggested and
chromatographic confirmation for one of these obtained. The in vitro
activity of 2-hydroxydesethylamodiaquine is shown to be 1% that of
amodiaquine for two chloroquine sensitive Plasmodiumfdcipamm strains
(43). The metabolism of 2-amino-4- quinoline derivatives of chloroquine
and amodiaquine in humans has been compared by Pussard et al. (41).
7.2

Pharmacokinetics

Amodiaquine hydrochloride is readily absorbed from gastro-intestinal


tract after oral administration, and higher concentrations occur in
erythrocytes, kidney, liver, lungs and spleen than in the plasma. After
absorption it is slowly released into the blood and excreted in the urine for
at least seven days after a single dose. The rate of excretion is increased in
acid urine (5,7). Amodiaquine is altered rapidly in vivo to yield products
which appear to be excreted slowly, and thus have a prolonged suppressive
activity (44).
Following a single oral dose of 10 mgkg of amodiaquine to five human
subjects, serum concentrations of 0.30 to 0.68 pg/ml (mean 0.5) have been
reported after four hours; the ratio of erythrocyte to serum concentration
varies with time and between individuals, but erythrocyte concentrations
are generally higher than the serum concentrations after forty-eight hours
(36).
The metabolic transformation of amodiaquine to monodesethylamodiaquine, and its pharmacokinetics in humans have been reported
(41).
8.

TOXICITY

Amodiaquine hydrochloride is an antimalarial of low toxicity and is


three to four times as active as quinine as a suppressive drug against
Plasmodium vivav and Plasmodium fakiparum infections (44,45,46). Jn
therapeutic doses amodiaquine hydrochloride is generally well tolerated
but may occasionally give rise to side-effects, including nausea, vomiting,

70

IQBAL AHMAD. TAUQIR AHMAD. AND K . USMANCHANI

diarrhoea, insomnia, vertigo, and lethargy (5).


The prolonged use of amodiaquine hydrochloride in the dosages
necessary to treat lupus erythematosus and rheumatoid arthritis is not
recommended, for corneal opacities and retinopathy, peripheral
neuropathy, fatal blood dyscrasias, and fatal hepatitis have been reported
after these large dosages (47). Patients have experienced involuntary
movements, usually with speech difficulty, after large but not excessive
doses of amodiaquine (48). It may cause birth defects if taken during
pregnancy (49).
A method is described for evaluating the relative toxicity of
amodiaquine in rats on the basis of effect on growth, lethal effects,
production of pathological changes, and the concentration of drug in blood
or plasma. The test can be completed in fourteen days (50).
ACKNOWLEDGEMENT

The authors wish to thank the United States Pharmacopeial


Convention, Inc., for donating a sample of amodiaquine hydrochloride.
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