Analysis 0f Chloroquine

‫ﺑﺴﻢ اﻟﻠﺔ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ‬
Analysis 0f Chloroquine Phosphate by

Derivative UV -Spectrophotometry
Submitted by:
Siham Abdoun Mohammed Abdel Alla
Supervised by:
Kamal Eldin Eltayeb Ibrahium
Department of Pharmaceutical Chemistry
University of Khartoum
Faculty of Pharmacy
April 2004
Acknowledgments
I would like to express my great thanks to professor Kamal Eldin
Eltayeb, head department of pharmaceutical chemistry, Faculty of
Pharmacy, University of Khartoum for great help in doing this work and
for providing the breadth and depth of his knowledge and experience and
for his careful supervision.
Also my thanks to professor Mohammed Alzein Haga, Faculty of
Pharmacy, Omdurman Islamic University for his great assistance and for
his helpful advice and guidance .
My great thanks extended to Balsam Pharmaceutical Company and to
Marrwa Pharmaceutical factory for the allowance to use their quality
control laboratories to do this work and to all the staff of these
laboratories for their great assistance. My great thank also to the Drug
Quality Control and Research Directorate and to Ministry of Health for
the financial support of this work.
I would also like to send my great thanks to all my entire colleagues and
friends for their assistance and support.
First and before all I thank God.

Contents
List of Figures V
List of Tables IX
Abstract English XV
Abstract Arabic XVIII
Acknowledgement XXI
1 Introduction
1.1. Malaria 1
1.1.1. Chemotherapy of malaria 2
1.1.2. Prophylaxis and suppression of malaria 7
1.1.3. Resistance 9
1.1.4. Treatment of malaria 10
1.1.5. Chloroquine 12
1.1.5.1. Origin 13
1.1.5.2. Physical Properties 13
1.1.5.3. pH 13
1.1.5.4. Solubility 14
1.1.5.5. Preparations of Chloroquine Salts 14
1.1.5.6. Uses and Administration 15
1.1.5.7. Absorption and Fate 17
1.1.5.8. Toxicity 18
1.2. Objectives of analysis 19
1.2.1. Spectrophotometry 20
1.2.1.1. Quantitative spectrophotometric assay of medicinal 21
substances
1.2.1.1.1. Assay of single components 23
1.2.1.1.2. Assay of multi-component samples 24
1.2.1.1.3. Assay-using absorbance corrected for interference 24
1.2.1.1.4. Assay after solvent extraction of the sample 25
1.2.1.2. Method for determining concentration 25
1.2.1.2.1. Simultaneous equation method 25
1.2.1.2.2. Absorbance ratio method 25
1.2.1.2.3. Geometric Correlation Method 26
1.2.1.2.4. Orthogonal Polynomial Method 26
1.2.1.2.5. Difference spectrophotometry 27
1.2.1.2.6. Colorimetric Measurements: 30
1.2.1.2.7. Derivative Spectrophotometry 31
1.2.1.2.8. chemical Derivatisation 35
1.2.2. Chromatographic techniques 36
1.2.2.1. High Performance Liquid Chromatography (HPLC) 37
1.3. Analysis of chloroquine 38
2 Objectives
3 Methodology
.3.1 Instrumentation 41
3.2. Reagents 41
3.3. Formulations selected to be analyzed 41
3.4. Stock Solutions of chloroquine standard 43
3.4.2. Stock solutions of excipent 43
3.5. Stock solutions pharmaceutical preparations (samples) 45
3.5.1. Stock solutions of chloroquine phosphate tablets 45
3.5.2. Stock solutions of chloroquine phosphate injection 45
3.5.3. Stock solutions of chloroquine phosphate syrup 45
3.6. Procedure 45
3.6.1. Spectrophotometric method 45
3.6.1.1. Calibration curve 45
3.6.1.2. Assay of chloroquine phosphate in presence of 46
excipent
3.6.1.3. Assay of chloroquine phosphate in pharmaceutical 47
dosage form
3.6.2. High Performance Liquid Chromatography Method 47
3.6.2.1. Phosphate buffer pH 3 47
3.6.2.2. Calibration curve 48
3.6.2.3. Assay of chloroquine phosphate in present of excipent 48
3.6.2.4. Assay of chloroquine phosphate in pharmaceutical 49
dosage forms
3.6.3. Non –Aqueous Titration Method 49
3.6.3.1 Assay of chloroquine phosphate tablets 49
3.6.3.2. Assay of chloroquine phosphate injection 50
3.6.3.3. Assay of chloroquine phosphate syrup 50
3.7. Application of derivative spectroscopy in analysis of 51
chloroquine phosphate in biological fluids
3.7.1. Calibration curve 51
3.7.2. Assay of chloroquine phosphate in pure plasma 51
4. Results of analysis
4.1. Calibration curves 53
4.1.1. Derivative spectrophotometry 54
4.1.2. Absorbance spectrophotometry 60
4.1.3. HPLC 66
4.2. Analysis of Chloroquine Phosphate in presence of 68
pharmaceutical excipent
4.3. Excipent Used In Chloroquine Phosphate Formulation 93
4.4. Analysis of chloroquine phosphate in pharmaceutical 101
dosage form
5. Discussion and Recommendation
5.1. Discussion 112
5.1.1. Derivative spectrophotometry calibration curves 113
5.1.2. Absorbance spectrophotometry calibration curves 114
5.1.3. Analysis of chloroquine phosphate in presence of 117
excipent
5.1.4. Analysis of chloroquine phosphate in pharmaceutical 120
dosage forms
5.1.5. Application of spectrophotometric derivative method 123
in the in vitro analysis of chloroquine phosphate in
biological fluids
5.2. Recommendation 127
Reference 128
Appendix 134
List of figure
Figure 1. Calibration graphs for derivative spectrophotometry analysis 55
of chloroquine phosphate standard in water at wavelengths
(225 nm, 239 nm, 260 nm, 333 nm and 349 nm)
of chloroquine phosphate standard in 0.01M HCl at
wavelengths (225 nm, 239 nm, 260 nm, 333 nm and 349 nm)
of chloroquine phosphate standard in plasma at wavelengths
(333 nm and 349 nm)
Figure 4. Calibration graphs for absorbance spectrophotometry analysis 61
of chloroquine phosphate standard in water at wavelengths
(221 nm, 236 nm, 256 nm, 331 nm and 343 nm)
of chloroquine phosphate standard in 0.01 M HCl at
wavelengths (221 nm, 236 nm, 256 nm, 331 nm and 343 nm)
of chloroquine phosphate standard in plasma at wavelengths
(331 nm and 343 nm)
Figure 7. Calibration graphs for high performance liquid 67
chromatography analysis of chloroquine phosphate standard
solutions
Figure 8. Results of analysis of chloroquine phosphate in presence of 70
starch by the three methods
aerosil by the three methods
avicel by the three methods
Figure 11 . Results of analysis of chloroquine phosphate in presence of 73
Mg sterate by the three methods
lactose by the three methods
talc by the three methods
acacia by the three methods
methylparaben by the three methods
propylparaben by the three methods
dicalcium phosphate by the three methods
titanium dioxide by the three methods
opadry by the three methods
propylene glycol by the three methods
glycerin by the three methods
sucrose by the three methods
saccharin sodium by the three methods
sodium citrate by the three methods
citric acid by the three methods
sorbitol by the three methods
xanthan gum by the three methods
tween 80 by the three methods
gelatin by the three methods
phosphoric acid by the three methods
Figure 31. Comparison of the results of analysis of chloroquine 94
phosphate in presence of disintegrant by the three methods
phosphate in presence of lubricant by the three methods
phosphate in presence of diluents by the three methods
phosphate in presence of coloring agents by the three methods
phosphate in presence of pH adjustment excipent by the three
methods
phosphate in presence of anti-microbial agents by the three
methods
phosphate in presence of emulsifying agents by the three
methods
Figure 38. Results of analysis of chloroquine phosphate injection by the 102
four methods
Figure 39. Results of analysis of balsaquine injection by the four 103
methods
Figure 40. Results of analysis of efroquine tablets by the four methods 104
Figure 41. Results of analysis of chloroquine phosphate tablets by the 105
four methods
Figure 42. Results of analysis of amiquine tablets by the four methods 106
Figure 43. Results of analysis of lariago tablets by the four methods 107
Figure 44. Results of analysis of chloroquine syrup (JPM) by the four 108
methods
Figure 45. Results of analysis of lariago syrup by the four methods 109
Figure 46. Results of analysis of chloroquine phosphate in pure plasma 111
by
the first derivative and zero order absorption spectroscopy
List of Tables
Table 1. Exciepent used in chloroquine phosphate formulations 44
Table 2. Derivative spectrophotometry results for analysis of 54
chloroquine phosphate standard in water
chloroquine standard phosphate in 0.01 M HCl
chloroquine phosphate standard in plasma
Table 5. Absorbance spectrophotometry results for analysis of 60
chloroquine phosphate standard in water
chloroquine phosphate standard in 0.01 M HCl
chloroquine phosphate standard in plasma
Table 8. High performance liquid chromatography results for analysis of 66
chloroquine phosphate standard
Table 9. Statistical analysis of the calibration graphs of chloroquine 114
phosphate using first derivative spectrophotometry
Table Statistical analysis of the calibration graphs of chloroquine 115
10. phosphate using zero order absorbance spectrophotometry
11. phosphate using first derivative, zero order absorbance
spectrophotometry and HPLC methods
12. phosphate standard in plasma using first derivative
Appendix
Table Results of analysis of chloroquine phosphate in presence of i
13. starch by the three methods
Table Statistical analysis of the results of chloroquine phosphate in i
14. presence of starch analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of ii
15. aerosil by the three methods
Table Statistical analysis of the results of chloroquine phosphate in ii
16. presence of aerosil analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of iii
17. avicel by the three methods
Table Statistical analysis of the results of chloroquine phosphate in ِiii
18. presence of avicel analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of Mg iv
19. sterate by the three methods
Table Statistical analysis of the results of chloroquine phosphate in iv
20. presence of Mg sterate analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of v
21. lactose by the three methods
Table Statistical analysis of the results of chloroquine phosphate in v
22. presence of lactose analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of talc vi
23. by the three methods
Table Statistical analysis of the results of chloroquine phosphate in vi
24. presence of talc analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of vii
25. accacia by the three methods
Table Statistical analysis of the results of chloroquine phosphate in vii
26. presence of accacia analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of viii
27. methyl paraben by the three methods
Table Statistical analysis of the results of chloroquine phosphate in viii
28. presence of methyl paraben analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of ix
29. propyl paraben by the three methods
Table Statistical analysis of the results of chloroquine phosphate in ix
30. presence of propyl paraben analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of x
31. dicalcium phosphate by the three methods
Table Statistical analysis of the results of chloroquine phosphate in x
32. presence of dicalcium phosphate analyzed by the three
methods
Table Results of analysis of chloroquine phosphate in presence of xi
33. titanium dioxide by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xi
34. presence of titanium dioxide analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xii
35. opadry by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xii
36. presence of opadry analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xiii
37. propylene glycol by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xiii
38. presence of propylene glycol analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xiv
39. glycerin by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xiv
40. presence of glycerin analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xv
41. sucrose by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xv
42. presence of sucrose analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xvi
43. saccharin sodium by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xvi
44. presence of saccharin sodium analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xvii
45. sodium citrate by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xvii
46. presence of sodium citrate analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xviii
47. citric acid by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xviii
48. presence of citric acid analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xix
49. sorbitol by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xix
50. presence of sorbitol analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xx
51. xanthan gum by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xx
52. presence of xanthan gum analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxi
53. tween 80 by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xxi
54. presence of tween 80 analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxii
55. gelatin by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xxii
56. presence of gelatin analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxiii
57. phosphoric acid by the three methods
Table Statistical analysis of the results of chloroquine phosphate in xxiii
58. presence of phosphoric acid analyzed by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxiv
59. disintegrants used in the formulations by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxiv
60. lubricant used in the formulations by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxv
61. diluents used in the formulations by the three methods
Table Results of analysis of chloroquine phosphate in presence of xxvi
62. coloring agents used in the formulations by the three methods
Results of analysis of chloroquine phosphate in presence of pH xxvi
Table adjustment agents used in the formulations by the three
63. methods
Table Results of analysis of chloroquine phosphate in presence of xxvii

64. anti-microbial agents used in the formulations by the three
methods
Table Results of analysis of chloroquine phosphate in presence of xxvii
65. emulsifying agents used in the formulations by the three
methods
Table Results of analysis of chloroquine phosphate injection by the xxviii
66. four methods
Table Statistical analysis of the results of chloroquine phosphate xxix

67. injection analyzed by the four methods
Table Results of analysis of basaquine injection by the four methods xxx
68.
Table 69 Statistical analysis of the results of basaquine injection xxx
analyzed by the four methods
Table Results of analysis of efroquine tablets by the four methods xxxi
70.
Table Statistical analysis of the results of efroquine tablets analyzed xxxi
71. by the four methods
Table Results of analysis of chloroquine phosphate tablets by the four xxxii
72. methods
Table Statistical analysis of the results of chloroquine phosphate xxxii
73. tablets analyzed by the four methods
Table Results of analysis of amiquine tablets by the four methods xxxiii
74.
Table Statistical analysis of the results of amiquine tablets analyzed xxxiii
75. by the four methods
Table Results of analysis of lariago tablets by the four methods xxxiv
76.
Table Statistical analysis of the results of lariago tablets analyzed by xxxiv
77. the four methods
Table Results of analysis of chloroquine syrup (JPM) by the four xxxv
78. methods
Table Statistical analysis of the results of chloroquine syrup (JPM) xxxv
79. analyzed by the four methods
Table Results of analysis of lariago syrup by the four methods xxxvi
80.
Table Statistical analysis of the results of lariago syrup analyzed by xxxvi
81. the four methods
Table Results of analysis of Chloroquine Phosphate standard in xxxvii
82. plasma
by the first derivative and zero absorption spectroscopy
1.1. Malaria:
Malaria is one of the most serious protozoal infectious diseases in
man. It is caused infection by the four species of Plasmodium. P.
falciprum causes falciprum (malignant tertian or sub tertian) malaria
which is the most serious form and can be fatal in non immune
individuals if not treated promptly. P. vivax causes vivax (benign
tertian) malaria which is widespread but rarely fatal, although
symptoms during the primary attack can be severe. P. malaria
causes quartan malaria which is generally mild but causes fatal
nephrosis. P. ovale causes ovale (ovale tertian ) malaria which is the
least common type, and produces clinical features similar to P. vivax
(James et al, 1989).
Malaria is a major threat to health and blocks the path to economic
development for individuals, communities and nations.
Almost half the world’s populations are at risk from this disease. It
causes one hundred million clinical cases and over one million
deaths each year. While over 80% of malaria cases and deaths occur
in Africa, malaria is a problem in every region of the world affecting
both young and old.

Children are particularly at risk, malaria being one of the childhood
killers in tropical Africa. It takes the lives of 1 out of 20 children
before the age of five. The disease causes anemia in children and
pregnant women. It affects the poor and under privileged most
severely.
Social, political and economical changes contribute much to the
worsening malaria problem, particularly through large scale
uncontrolled population movements and ecological disturbances
(James et al, 1989).
Construction and environmental changes brought about by
development are creating environment favorable for malaria
transmission. This greatly enhance exacerbating existing problems
opening the way for devastating epidemics in areas which are
previously malaria-free, leading to many deaths and impoverishment
of communities (Bruce-Chwatts et al, 1989).
In Sudan, malaria is an endemic disease all over the country with varying
degrees from holodemisty in the South to hypodemisty in the North. It
is the most serious public health hazard causing high morbidity and
mortality which adversely affects the socio-economic development. The

disease in Sudan is mostly due to infection with P. falciprum (Roll I.M,
1986).
1.1.1. Chemotherapy of malaria:
Antimalarial drugs are classified according to the stage of parasitic
life cycle, which they affect.
Blood schizotocides act on the erythrocytic stages of the parasite,
which are directly responsible for the clinical symptoms of the
disease. They produce clinical cure or suppression of infection by
susceptible strains of all the four species of malaria parasite.
However, since they have no effect on exoerythrocytic forms, they
do not produce a radical cure of relapsing forms of ovale or vivax
malarias.
Tissue schizotocides act on exoerythrocytic stages of the parasite
and have been used for prophylaxis to prevent invasion of the blood
cells, or as anti relapse drugs to produce radical cure of vivax and
ovale malaria.
Gametocyticides destroy the sexual forms of the parasite to interrupt
transmission of the infection to the mosquito vector.
Sporonticides have no direct effect on the gametocytes in the human
host but prevent sporogony in the mosquito.

The antimalarial drugs are principally classified by chemical
structure (Susan Budavari et al, 1996) into:
1.1.1.1. quinine salts, which are rapidly acting, blood schizontocides
with some gametocidal activity. They have the chemical structure:
H C H
2
H O N
H 3C O H
N
(8 α , 9 R) – 6 – Methoxycinchonan – 9 – ol (C20H24N2O2)
Qinine is available as the following salt:
1.Quinine bisulfate heptahydrate (C20H24N2O2 .H2SO4.7 H2O).
2. Quinine dihydrochloride (C20H24N2O2.2HCl).
3. Quinine hydrochloride dihydrate (C20H24N2O2.HCl.2H2O).
4. Quinine sulfate dihydrate (C20H24N2O2)2 H2SO4.2H2O.
1.1.1.2. The Quinoline: Has the chemical strucure:
benzo [6] pyridine (C9H7N)
Derivatives of the Quinoline, which has antimalarial activities, are:
1.1.1.2.1. 8-aminoquinolines: they are tissue schizontocides mainly
used to prevent relapse of ovale and vivax malaria, and have

gametocytcidal activity. They also have some activity at other stages
of the parasites life cycle. Example is primaquine and quinocide.
The quinocide has the chemical structure:
C H3
HN
N NH
2
H3CO
8 – [(4 – amino – methyl butyl) amino] – 6 – methoxy
quinoline(C15H21N3O)
It is available as:
1. Quinocide diphosphate (C15H27N3O9P2).
2. Quinocide hydrochloride (C15H21N3O.HCl).
3. Quinocide dihydrochloride (C15H21N3O.2 HCl).
The chemical structure of primaquine is:

H
H3C O N
N H2 . 2 H3P O 4
C H3
N
( ± ) –8–[(4–Amino–1– methyl butyl) amino] –6–methoxy quinoline
phosphate (1:2) (C15H21N3O .2H3PO4)
1.1.1.2.2. 4-aminoquinolines: they are rapidly acting blood
schizontocides with some gametocidal activity. Examples are
amodiaquine and chloroquine, which has the chemical structure:

Cl N
HN
N
C H3
C H3 C H3
7 – chloro – 4 –(4– diethyl amino–1–methyl butyl amino)quinoline
(C18H26Cl N3)
1.1.1.3. 9-aminoacridines such as mepacrine, which is a blood
schizontocide with some gametocidal activity. It has the following
structure:
NH2
N
C13H10N2
It is available as hydrochloride (C13H10N2.HCl)
1.1.1.4. biguanides, such as proguanil, having dihyrofolate reductase
inhibitor activity. They are tissue schizontocides used mainly for
prophylaxis of falciprum malaria. They also have some activity
against blood schizonts. It is chemical structure:
NH NH
H2N N N H2
H
Amino guanidine (C2H7N5)
1.1.1.5. diaminopyrimidines such as pyrimethamine, have an action
similar to that of the biguanides. The chemical structure of the
pyrimethamine is
N NH2
H3C
N
NH2
Cl
2,4 – diamino –5–(p– chlorophenyl) – 6 – ethylpyrimidine
(C12H13ClN4)
1.1.1.6.The sesquiterpine lactones such as artemisin and artemether,
which act mainly as blood schizontocides. The artemether has the
chemical structure:
CH3
H
O O
H 3C O H
H O H
H OCH
O C H3 3
Dihydro artemisinin methyl ether (C16H26O5 )
While the artemisin has the structure:

C H3
O H
H
O C H3
H
C H3 O
6 α 8 α –dihydroxy–3 – oxoeudesma–1,4–dien 12–oic acid 12, 6–
lactone
C15H18O4
Other drugs with anti malarial activity include the sulphonamides,
mefloquine, dapsone and some antibiotics such as tetracycline and
erythromycin (James et, al 1989).
1.1.2. Prophylaxis and suppression of malaria:
The increasing prevalence of drug resistant strains particularly of P.
falciprum has made recommendations for malaria prophylaxis
difficult. The WHO believes that the widespread use of
chemoprophylaxis in immune or semi-immune population is no
longer recommended. Chemoprophylaxis as malaria control strategy
should aim at lowering morbidity levels in groups that are at high
risk from sever and complicated malaria, notably pregnant women
and non- immune visitors. Since the prophylaxis afforded by many
chemoprophylactic regimens is not absolute, travelers should be
advised on methods of avoiding mosquito’s bites.
Chemoprophylaxis may be (causal prophylaxis) referring to absolute
prevention of infection or (clinical prophylaxis) i.e. suppression of
parasitaemia and it is symptoms. Causal prophylaxis is provided by
tissue schizontocides, which destroy the erythrocytic form of the
parasite. Blood schizontocides produce suppression or clinical

prophylaxis, which if continued until all erythrocytic parasites are
destroyed, will ultimately produce a suppressive cure.
Recommendation on chemoprophylaxis must be based on the basis
of the occurrence and susceptibility of malaria strains in the
particular geographical area. Local variations due to altitude, rainfall
and seasonal differences in temperature, further complicate the
matter. The toxicity of some chemoprophylaxis regimens has further
worsened the situation since the incident of toxic reaction can
approach or exceed the danger of contracting malaria.
In general, amodiaquine should not be used for prophylaxis. Further
more many experts, including the WHO, also agree that a
combination of pyrimethamine with sulphadoxine (fansidar) should
not be used for prophylaxis.
In areas of little or no chloroquine resistance, chloroquine may still
be used. However some experts suggest proguanil as an alternative.
In areas of low-grade chloroquine resistance, chloroquine may still
alleviate the infection. In areas with high –grade chloroquine
resistance a combination of chloroquine and fansidar were used to be
recommended in the past. This is now considered to be too toxic and
in any case multidrug- resistant strains of malaria are increasingly

widespread. Chloroquine plus pyrimethamine-dapsone is a possible
alternative but the potential high toxicity is once again a problem.
Less toxic regimens should be considered even if they are less
effective. Mefloquine may be useful, if available, but there is
concern that indiscriminate prophylactic use may induce resistance
to mefloquine (James et al 1989).
1.1.3. Resistance:
Resistance of P. falciprum to most of the current anti malarial drugs
has stemmed as a main technical problem in malaria control. Drug
resistance has being defined as the ability of parasite strains to
multiply and or to survive in the presence of concentrations of a drug
that normally destroys parasite of the same species or prevent their
multiplication. Although drug resistance has also been observed in
other plasmodium species, it is the resistance of p. falciprum and in
particular to chloroquine, which has drawn most attention. The
WHO has developed an arbitrary grading system to describe the

relative degree of resistance of strains of P. falciprum to
chloroquine.
Sensitivity is described as the clearance of asexual parasitaemia within 7
days of initiation of treatment, without subsequent recrudescence.
Chloroquine resistance developed at almost the same time in
Southern Asia and in South America at the end of the 1950s.
Resistance now affects most of Asia and the Western Pacific Islands
with evidence of westward spread. It is well established in South and
Central America. Major and alarming changes in susceptibility have
occurred in Africa, south of Sahara and there is now evidence of
chloroquine resistance in West Africa.
A knowledge of the extent of resistance in terms of geographical
distribution and degree of resistance is important for the selection of
appropriate control measures and for development of policies for
rational use of antimalarial drugs. The use of sub curative doses o f
drugs contributes to resistance. Radical curative treatment is
probably the most reliable way of avoiding selective parasite
survival. Mass drug administration for suppression should therefore
be avoided.
The use of combination of drugs of similar half –life may also delay
the emergence of resistant strains (James et al, 1989).
1.1.4. Treatment of malaria :
Early and proper diagnosis and prompt treatment is the most
important component of the Global Malaria Control Strategy.
Effective treatment should be made to all persons at risk, and also
forms the basis of malaria control interventions.
Severe malaria is a medical emergency requiring immediate
administration of rapidly effective anti malaria drugs. When
possible, drug should be given intravenously, but studies suggest
adequate absorption of quinine, artesunate, and chloroquine after
intramuscular injections, which are acceptable alternatives.
Parentral artemether is given only by intramuscular injection.
Suppositories of chloroquine, artemisin, and artesunate are available
in some countries but more pharmacokinetic data are needed for
these drugs. Quinine and Quinimax should be administered
intrarectally. Parentral treatment is needed in acute stages but oral
treatment should be substituted as soon as the patient is able to take
fluids by mouth.
In cases, where it is not possible to give parentral treatment,
administration by mouth or via nasogatric tube should be started
immediately as drug absorption is believed to be adequate even in
sever malaria.
Chloroquine is still the drug of choice for treatment of
uncomplicated malaria in Sudan. It is safe, of affordable price and
has been used over a long time for treatment of malaria. It is noted,
however, that many patients do not respond to the treatment with
chloroquine (Mahmoud et. al, 1988). This lead to consider that the
parasite has developed resistance against chloroquine in certain
regions in Sudan (Bayoumi et. al, 1988). The increasing number of
patients showing resistance to therapeutic treatment with
chloroquine led to questioning the drug efficacy, before deciding to
change the drug policy on the first –line antimalarial drug. The
extent and nature of true drug –resistance should be clarified
whether due to substandard drug or to other reasons.
The supply and availability of drugs in the country have been
increased substantially over the last ten years. Many product-
formulations of chloroquine from different sources have been
introduced into the market.

However, the detection of substandard and counterfeit drugs within
distribution channels in some developing countries, has led to great
concern about the quality of the drugs. In addition, the tropical
climatic conditions i.e. high temperature and humidity may affect the
quality and stability of the drug and thus may lead to physical
deterioration or chemical decomposition resulting in reduced
activity.
The stability of the drug depends largely upon its formulation, thus
its expiry date should be determined on the basis of stability studies.
Thus it is significant to determine the expiry date on the label of the
product.
Toward the end of this study the ministry of health introduce in

the market new regimens for treating malaria (artesunate and
fansidar) and (artesunate and
lumfantrene). Chloroquine will be withdrawn gradually and replaced
by these
regimens in chloroquine resistance regions.

1.1.5. Chloroquine:
Chloroquine, (British approved name, 4- aminoquinoline derivative),
has the chemical structure shown in 1.1.1.2.2.
It is available as the following salts:
1. Chloroquine hydrochloride (BANM) C18H26ClN3, 2HCl

2. Chloroquine phosphate C18H26ClN3, 2H3PO4
3. Chloroquine sulphate C18H26ClN3, H2SO4, H2O
It is official in the British pharmacopoeia as Chloroquine sulphate
and Chloroquine phosphate.
In the United State Pharmacopoeia it is found as Chloroquine and
Chloroquine phosphate.
1.1.5.1. Origin:
Chloroquine was originally synthesized in 1934 by Bayer in Germany
(Resochin); it is prepared by the condensation of 4-7-dichloro quinolone
with 1-diethylamino-4-amino pentane.
1.1.5.2. Physical Properties:
Chloroquine base is a white or slightly yellow, odorless, crystalline
powder with a bitter taste, with a melting range of 870 C - 920C.
Chloroquine phosphate is a white or almost white, odorless,
crystalline powder with a bitter taste and it is hygroscopic. It is
exists in two polymorphic forms, one form melt in the range of 1930
C - 1950C and the other in the range of 2100 C - 2180C.
Chloroquine sulphate is a white or almost white, odorless, crystalline
powder, melting in the range 2050 C - 2100C.

1.1.5.3. pH:
10%w/v solution of chloroquine phosphate has a pH of 3.8 to 4.3.
8%w/v solution of chloroquine sulphate has a pH of 4 to 5.
1% w/v solution of chloroquine phosphate has a pH of about 4.5.
1.1.5.4. Solubility:
Chloroquine is very slightly soluble in water, soluble in chloroform,
in ether and dilute acids.
Chloroquine phosphate is freely soluble (1 in 4) in water, although
solubility is reduced at alkaline or neutral pH. It is practically
insoluble in ethanol and benzene, very slightly soluble in
chloroform, ether and methanol.
Chloroquine sulphate is soluble (1 in 3) in water and very slightly
soluble in ethanol, freely soluble in methanol, practically insoluble
in acetone, chloroform and ether (Walter Lund, 1994) (15).
1.1.5.5. Preparations of Chloroquine Salts:

1. Chloroquine hydrochloride injection (U.S.P.)
A sterile solution of chloroquine in water for injection prepared with aid
of hydrochloric acid; it contains 47.5 to 52.5 mg ml-1of C18H26ClN3,
2HCl, equivalent to approximately 38.7 to 42.8 mg of chloroquine base,
pH 5.5 to 6.5.
2. Chloroquine phosphate injection (B.P.)
It is potency is expressed in terms of the equivalent amount of
chloroquine base.
3. Chloroquine sulphate injection (B.P.): It is potency is expressed in
terms of equivalent amount of chloroquine base, pH 4.0 - 5.5.
4. Chloroquine phosphate tablets (B.P.)
5. Chloroquine phosphate tablets (U.S. P.)
6. Chloroquine sulphate tablets (B.P.)
1.1.5.6. Uses and Administration:
Chloroquine is used for the suppression and clinical cure of malaria
due to susceptible strains of P. falciprum, P. ovale, P. vivax, and P.
malaria. It is a rapidly acting blood schizontocide with some
gametocyticidal activity against P. ovale, P. vivax, and P. malaria
and immature gametocytes of P. falciprum. Chloroquine does not
produce a radical cure of vivax or ovale malarias since it has no

activity against exoerythrocytic forms. The mechanism of action of
chloroquine against blood schizonts is still not clear but it appears to
act by influencing hemoglobin digestion by the malaria parasite. It
also interferes with synthesis of nucleoproteins by the parasite
(James et. al, 1989).
In addition to it is antimalarial action, chloroquine has been used in
the treatment of amoebic hepatitis in conjunction with emetine or
dehydroemetine but it has no effect on intestinal amoebiasis.
Chloroquine has also been used in rheumatoid arthritis.
However this is limited by it is toxicity particularly when it
is administered for a long period.
Chloroquine is given as phosphate, sulphate or hydrochloride. Doses
are normally expressed in terms of chloroquine base 300 mg, which
is approximately equivalent to 500 mg of chloroquine phosphate or
400 mg of chloroquine sulphate. 40 mg chloroquine base is
approximately equivalent to 50 mg of chloroquine hydrochloride.
For the treatment of acute attack of malaria the usual oral dose is 600
mg chloroquine base initially, then 300 mg after 6 hours, then 300
mg daily on each of the two following days giving a total dose of
approximately 25 mg per kg body- weight.

Traditionally a single dose of 10 mg per kg has been used to treat
malaria in semi-immune subjects. The WHO now recommends that
all patients be given the full course of 25 mg per kg over 3 days in
attempts to slow spread of the drug resistance. A recommended dose
for treatment in children is 10 mg per kg, followed by 5 mg per kg
after 6 hours, then 5 mg per kg daily for 2 days. In sever and
complicated malaria, when the patient is unable to take oral
medication, parenteral chloroquine may be used. However, many
authorities now recommend the use of quinine instead of
chloroquine in view of the serious toxicity, which may occur
following parentral administration of chloroquine.
There is also some dispute about the most appropriate route for
chloroquine injection: some favour intramuscular injection at a dose
of 3 mg of base per kg every 6 to 8 hours to a maximum of 1 g over
24 hours. Others prefer to give chloroquine by slow intravenous
infusion of 5 mg of base per kg in 10 ml of infusion fluid per kg
given over 4 hours and repeated at 12-hourly intervals. The sever
toxicity which is encountered after parentral administration is
probably due to the very high blood concentrations which follow
both intravenous and intramuscular administration, but

administration as an infusion over 4 hours may help to minimize
this. Parentral administration of chloroquine should only be
undertaken while the blood pressure is carefully monitored and
should be replaced by oral administration as soon as possible. The
situation in parentral administration is even more difficult in
children since there is evidence that they are particularly susceptible
to the adverse cardiovascular effects of chloroquine.
In many countries where chloroquine is freely available, it may be
difficult to discover whether chloroquine has been taken recently and
therefore unexpected toxicity can result during subsequent parentral
administration (James et. al, 1989).
1.1.5.7. Absorption and Fate:
Chloroquine is readily absorbed from the gastro-intestinal tract and
about 55% in the circulation is bound to plasma proteins. It
accumulates in high concentrations in some tissues such as the
kidneys, liver, lung and spleen. It is strongly bound in melanin –
containing cells such as those in eyes and skin. Chloroquine is
eliminated very slowly from the body and it may persist in tissues
for a prolonged period.

The drug is excreted mainly in the urine, with 70% as unchanged
drug and about 25% as the desethyl metabolite. The rate of urinary
excretion of chloroquine is increased at low pH values.
Intramuscular and subcutaneous routes of administration of
chloroquine give almost identical plasma or whole blood
concentration profiles. Absorption is very rapid and relatively
complete even in sever infections. Chloroquine administered as
rectal suppositories has a bioavailability of approximately 30 % -50
%, and rate of absorption similar to that of oral chloroquine in
healthy volunteers.
1.1.5.8. Toxicity:
Chloroquine is one of the most widely used drugs in the world. It still
accounts for over 90% by weight of the global antimalarial drugs
consumption. It has undoubtedly saved the lives of many patients with
sever malaria.
Oral chloroquine is usually well tolerated. Nausea, headache, and
dysphoria are relatively common but seldom serious. Patients may vomit
and may complain of blurred vision. Postural hypotension associated
with malaria may be exacerbated and pruritus can be severe especially in
dark-skinned patients.
There are several reports of sudden death following administration of
intramuscular chloroquine to children with sever malaria. These
probably resulted from cardiovascular toxicity. Fatal hypotension would
be most likely if severely ill, febrile, dehydrated children were
inadvertently given a large intramuscular dose without allowing for
weight or surface area or if the injection was given intravenously by
mistake and the child was then nursed upright. This potentially lethal
toxicity is circumvented by giving continuous constant –rate intravenous
infusion, or smaller, more frequent intramuscular or subcutaneous doses
than previously recommended.
Chloroquine overdose is manifested by coma, convulsions,
dysrhythmias and hypotension.
Administration of more than 100 g of chloroquine (i.e. weekly
prophylactic use for more than 5 years) to adults is associated with
an increased risk of retinopathy. This appears to be associated
mainly with the higher doses used in collagen –vascular diseases.
All subjects on long- term chloroquine prophylaxis should have
regular ophthalmological examinations.
Skeletal and cardiac myopathy may occur in patients receiving high-
dose chloroquine maintance therapy for rheumatoid arthritis.

1.2. Objectives of analysis in quality control:
Analytical chemistry is concerned with the chemical characterization
of matter. It plays an important role in all aspects of chemistry e.g.
agricultural, clinical, forensic, environmental, manufacturing and
pharmaceutical chemistry.
The quality of manufactured products depends on the proper
chemical proportions, and measurement of constituents is thus a
necessary part of quality control.
The field of analytical chemistry consists of qualitative analysis and
quantitative analysis. The former deals with identification while the
later deals with determination of how much of one or more
constituents are present.
Qualitative tests may be performed by selective chemical reactions
or with the use of instrumentation like gas chromatography and mass
spectrometry. In case of quantitative analysis a history of the sample
composition should be known or the analyst must first do qualitative
tests.
There are three phases in analysis, which are the fast screen phase,
the identification phase, and the possible quantification. Many

techniques are used in the fast screen phase including gas
chromatography, liquid chromatography, and immunoassay.
There are many quantitative techniques including spectrophotometry
(ultraviolet, visible and infrared), fluorimetry, atomic spectroscopy
(absorption and emission), mass spectrometry, nuclear magnetic
resonance spectrometry (NMR), X-ray spectroscopy (absorption,
fluorescence), electro analytical techniques (potentiometry,
voltametry and electrolysis), chromatography techniques (liquid and
gas), gravimetry, titrimetry, and radiochemistry. Different
techniques possess varying degrees of selectivity, sensitivity,
precision, accuracy, cost and speed (Gray Christian, 1998).
1.2.1. Spectrophotometry:
The technique of ultra violet-visible spectrophotometry is one of the
most commonly used techniques in pharmaceutical and biomedical
analysis. It is employed in quantitative purposes and with certain
limitations for characterization of drugs, impurities, metabolites and
related substances. The technique basically involves the
measurement of the amount of ultraviolet (190-380 nm) or visible
(380-800 nm) radiation absorbed by a substance in solution.

Absorption of light in both the UV and visible region of the
electromagnetic spectrum occurs when the energy of the light
matches the energy required to induce an electronic transition and it
is associated vibration and rotational transsion in the molecule.
Spectral selectivity can be induced and/or enhanced by a number of
chemicals or by instrumental technique such as difference, higher-
derivative and dual wavelength spectrophotometry.
Spectral interference can often arise from what is known as
“irrelevant” nonspecific absorption, and also from absorption by
other materials and impurities, which may be present.
When interference is due to spectral overlap of two or more well-
defined components, a number of methods are adopted to measure
the individual concentrations. These methods include the vierordt
multiwavelength technique, least squares deconvolution and second
or higher derivative spectrophotometry (Moffat et. al, 1986).
1.2.1.1. Quantitative spectrophotometic assay of medicinal
substances:
The assay of an absorbing substance is carried out by making a
solution of the substance in a transparent solvent and measuring its
absorbance at a suitable wavelength. The wavelength normally

selected is a wavelength of maximum absorption ( λ max) where small
errors in the settings of the wavelength scale will have little effects
on the measured absorbance.
Ideally, the concentration should be adjusted to give an absorbance
reading of approximately 0.9, around which the accuracy and
precision of measurement are optimal. It is often preferred to read
the absorbance from the instrument display under non-scanning
conditions, i.e. with the monochromator set at the analytical
wavelength. Alternatively, the absorbance can be read from a
recorded spectrum of the substance obtained by using a recording
double-beam spectrophotometer. The later procedure is of particular
usefulness for qualitative purposes and in certain assays in which
absorbance at more than one wavelength are required. The
concentration of the absorbing substance can be calculated from the
measured absorbance using one of three principal procedures.
1. Use of standard absorbtivity value
This procedure is adopted by official compendia, e.g. British
Pharmacopoeia for stable substance.
2. Use of calibration graphs

In this procedure the absorbances of a number of standard solutions
(4-6) of reference substance at concentrations encompassing the
sample concentration are measured and a calibration graph is
constructed. Concentration of analyte can then be read from the
resultant graph.
3. Single-or double point standardization
The single point procedure involves the measurement of the
absorbance of a sample solution and of a standard solution of the
reference substance. The standard and sample solutions are prepared
in a similar manner; final concentration of the sample solution
should be close to that of the standard solution.
The concentration of the substance in the sample can be calculated
from proportional relationship that exists between absorbance and
concentration
A test XC std
Ctest = Where;
A std
Ctest is the concentration of test substance; Atest is absorbance of test
substance.
Cstd is the concentration of standard; Astd is absorbance of standard.

Pharmacopoeial applications include assays for single and mixtures
of drugs analysis involving color reactions (colorimetric methods),
test for tablet dissolution, limit test for impurities, and assays of bulk
drugs. Further applications are for physicochemical measurements,
such as pKa or velocity constants in enzymatic reactions .The scope
of such applications has been significantly extended by methods,
which can confer additional specificity, namely difference
spectrophotometry, derivative spectophotometry, simultaneous
equation method, absorbance ratio method, geometric correction
method and orthogonal method.
1.2.1.1.1. Assay of single components:
When only one component in the sample absorbs significantly, the
wavelength is chosen to coincide with the center of a broad
maximum in the spectrum in order to minimize errors due to
wavelength –setting. In cases where the spectrum has no suitable
maximum, a flat absorption minimum can be used, provided that the
expected consequent loss of sensitivity is acceptable. Wavelengths
near the limits of the ultraviolet and visible ranges must be avoided,
because of the possibilities of stray-light errors.

The concentration of a component in a sample, which in addition
contains other absorbing substances, may be determined by a simple
spectrophotometric measurement. This is possible only if the other
components have a small absorbance at the wavelength of
measurement. This condition will be met if the concentration of the
interfering substances, their absorbtivity or the path length of the
solution is sufficiently small that their product can be ignored. An
example of this method is the assay of paracetamol in paediatric
paracetamol elixir (Beckett A. H and Stenlake J. B., 1997).
1.2.1.1.2. Assay of multi-component samples:
The spectrophotometric assay of drugs often involves the
measurement of absorbance of samples containing more than one
absorbing component.
A number of modifications to the simple spectrophotometric
procedure have been introduced, which may eliminate certain
sources of interference and permit the accurate determination of one
or all of the absorbing components.
Each modification of the basic procedure may be applied if certain
criteria are fulfilled. The basis of all the spectrophotometric

techniques for multi-component analysis of samples is the property
that at all wavelengths, the following facts are observed:
1. The absorbance of solution is the sum of the absorbencies of the
individual components.
2. The measured absorbance is the difference between the total
absorbance of the solution in the sample cell and that of the solution
in the reference cell (blank).
1.2.1.1.3. Assay using absorbance corrected for interference:
If the nature, concentration and absorptivity of the absorbing
interferents are known, it will be possible to calculate their
contribution to the total absorbance of a mixture. The concentration
of the absorbing component of interest can then be calculated from
the corrected absorbance (total absorbance minus the absorbance of
interfering substances).
1.2.1.1.4. Assay after solvent extraction of the sample:
If interference resulting from other absorbing substances is very
large or if its contribution to the total absorbance cannot be
calculated, it may be required, if possible, to separate the absorbing
interferents from the analyte by solvent extraction procedures. These
procedures are appropriate especially for acidic or basic drugs where

the state of ionization determines their solvent partitioning behavior.
Such an example is the assay of caffeine in aspirin and caffeine
tablets (B.P.).
1.2.1.2. Methods for determining concentration:
1.2.1.2.1. Simultaneous equation method:
If a sample contains two absorbing drugs each of which absorbs at
the λ max of the other, it may be possible to determine both drugs by
applying the technique of simultaneous equations (Vireod’s method)
provided that certain criteria are fullfield. An example of such assay
is described in the B.P. for the assay of quinine related alkaloids and
for cinchona-related alkaloids in Cinchona Bark.
1.2.1.2.2. A BSORBANCE RATIO METHOD
The method of absorbance ratio is a modification of the
simultaneous equation procedure. It depends on the property that, for
a substance, which obeys Beers Law at all wavelengths, the ratio of
absorbance at any two wavelengths is a constant value independent
of concentration or path length. In the quantitative assay of two
components in admixture by the absorbance ratio method,
measurement of absorbance are made at two wavelengths one being

the λ max of one of the components ( λ 2 ) and the other one being a
wavelength of equal absorbtivity of the two components ( λ1 ), i.e., an
iso-absorptive point. This method is applied for the assay of
trimethoprim and sulphmethoxazole in co-trimoxazole tablets
(Beckett A. H and Stenlake J. B., 1997).
1.2.1.2.3. G EOMETRIC CORRECTION METHOD
Mathematical correction procedures have been developed to reduce
or eliminate the background irrelevant absorption that may be
present in samples of biological nature. The simplest of these
procedures is the three –point geometric procedure, which is
applicable if the irrelevant absorption is linear at the three
wavelength selected. The assay of vitamin A in fish liver oils
furnishes an example for the geometric correction method (Beckett
A. H and Stenlake J. B., 1997).
1.2.1.2.4. Orthogonal polynomial method
The technique of orthogonal polynomials is another mathematical
correction procedure, which involves more complex calculations
than the three –point correction procedure. The basis of the method
is that an absorption spectrum may be represented in term of
orthogonal function as follows:

A ( λ ) = p0P0 ( λ ) + p1P1 ( λ ) + p2P2 ( λ )…..pnPn ( λ )
Where A is for the absorbance at wavelength λ belonging to a set
of n+1 equally spaced wavelengths at which the orthogonal
polynomials P0 ( λ ), P1 ( λ ), p2P2 ( λ )… pnPn ( λ ) are each
defined.
This method is applied in the selective assay of phenobarbitone,
combined with phenytoin in a capsule formulation (Beckett A. H
and Stenlake J. B., 1997).
1.2.1.2.5. Difference spectrophotometry:
Both selectivity and accuracy of spectrophotometric analysis of
samples, which, contain absorbing interferents, may be greatly
improved by the technique of difference spectrophotometry. In
difference spectrophotometric assays the measured value is the
difference in absorbance ( ∆A ) between two equimolar solutions of
the analyte, in different chemical forms, which exhibit different
spectral characteristics.
Difference spectrophotometry is a technique of compensating for the
extraneous materials present in a sample, which might interfere with
the spectrum of the drug being determined. It involves the
measurement of difference in the absorbance, at a defined
wavelength, between two samples in one of which a physical or
chemical property of the drug has been changed. It is assumed that
the spectrum of the drug can be changed without affecting the
spectrum of the interfering material. Alternatively, the absorbance
difference between the sample and an equivalent solution without
the drug may be measured. Difference spectrophotometry is
sometimes referred to as 'differential spectrophotometry', but this is
not recommended since it might be confused with derivative
spectrophotometry.
Certain criteria are required for applying difference
spectrophotometry for the analysis of a substance in the presence of
other absorbing substances:
1. Reproducible changes are induced in the spectrum of the analyte
by the addition of one or more reagents.
2. The absorbance of the interfering substances has not been altered
by the addition of such reagents.

The simplest and most commonly used technique for altering the
spectral properties of the analyte is the adjustment of the pH of the
solution by means of aqueous solution of acids, alkalis or buffers.
A substance whose spectrum is not affected by changes in pH can
also be determined by a difference spectrophotometric procedure.
This can be done by quantitatively converting it, using a suitable
reagent, to a chemical species that has different spectral properties
from the uncreated parent substance. The ∆A between equimolar
solutions of the uncreated substance and the corresponding
derivative is free of interference if the irrelevant absorption is
unaffected by the reagent.
If: ∆A = ∆ε bc
Where: ε = molar absorptivity (liter/mole/cm),
b = cell path-length (cm),
c = concentration (mol/liter).
Then the difference absorbance can be related to concentration by
the relation:
∆Atest/∆Astandard = Ctest/Cstandard
It should, however, be established that ∆A is a liner function of
concentration C
Difference spectrophotometry can be used for quality control if the
interfering material is well –defined. In such a case an appropriate
dilution of a suitable reference solution can be used in the reference
cell. The technique of difference absorbance is subject to systematic
error when there is uncertainty about the concentration of interfering
materials in the samples to be assayed. This error increases in
proportion to the ratio of the molar absorptivity of the interferent to
that of the analysed drug.
A further technique to correct for absorptive interferences by
difference measurement is based on dual –wavelength
spectrophotometry. In this method, two monochromatic beams at
different wavelengths are passed through the same sample. One
wavelength (λ1) is generally characteristic of the drug, while the
other (λ2) is carefully selected so that the absorbance is equivalent to
the level of absorptive interference (Aλ1m) anticipated at the
analytical wavelength (λ1). The second radiation beam will thus be
analogous to the reference cell employed in conventional difference
spectrophotometry. The difference in absorbance at the two

λ
wavelengths ( ∆ A) represents the absorption of drug (A n1 ) corrected
for interference
λ1 λ λ1
A =A n1 + A m
And since
λ2 λ1
A = Am
λ1 λ2 λ
Then ∆A =A -A = A n1
An application of this method is represented by the correction of
Raleigh scatter in samples of biological origin. An example of this
method is the selective assay of chlordiazepoxide and it is major
hydrolysis product, demoxepam, in degraded formulations of
chlordiazepoxide (Beckett A. H and Stenlake J. B., 1997).
1.2.1.2.6. Colorimetric Measurements:
In colorimetric methods we can selectively transform a drug, its
impurity, or a metabolite so that the spectrum of the product is
shifted to the visible region. This will minimize interference caused
by another drug, formulation components or biological substances,
thereby conferring a further degree of specificity. Moreover, a drug
with little or no useful absorption can be more highly – absorptive
chromophore.
There are several parameters, which require careful and critical
consideration in colormetry. Firstly, the colour reagent should be

selective for the drug molecule itself, discriminating against
degradation products, impurities, and formulation excipent which
might be present. Secondly, the effect and control of any parameters,
which is likely to affect the color reaction, should be established, i.e.
solvent, pH, temperature, reagent excess, order of mixing reagents,
and other related factors.
Moreover, the time required to establish the chromophore
generated, should be carefully monitored and assessed. Finally, the
analytical performance should be assessed in terms of recovery,
precision, sensitivity, linear range, and robust behavior.
1.2.1.2.7. DERIVATIVE SPECTROPHOTOMETRY
IN DERIVATIVE SPECTROPHOTOMETRY THE
ABSORBANCE (A) OF A SAMPLE IS DIFFERENTIATED WITH
RESPECT TO WAVELENGTH ( λ ) TO GENERATE THE FIRST,
SECOND, OR HIGHER ORDER DERIVATIVES (14). IN THE
CONTEXT OF DERIVATIVE SPECTROPHOTOMETRY, THE
NORMAL ABSORPTION SPECTRUM IS REFERRED TO AS
THE FUNDAMENTAL, ZERO ORDER OR 0D SPECTRUM
A= f ( λ )
2
dA dλ = f ' ( λ ) d 2 A dλ = f " ( λ ), etc.
Zero order first derivative second
derivatives
THE FIRST DERIVATIVE (1D) SPECTRUM IS A PLOT OF THE
RATE OF CHANGE OF ABSORBANCE WITH WAVELENGTH
AGAINST WAVELENGTH, I.E. A PLOT OF THE SLOPE OF
THE FUNDAMENTAL SPECTRUM AGAINST WAVELENGTH
OR A PLOT OF
dA dλ VS. λ .
The second derivative (2D) spectrum is a plot of the curvature of the

0
D spectrum against wavelength or a plot of d 2 A dλ
2
vs. λ .
Spectra are seen as distinctive bipolar feature; the even order
derivatives are bipolar functions of alternating sign at the centroid,
whose position coincides with that of the original peak maximum
(Moffat et. al,1986 ).
The first derivative spectrum of an absorption band is characterized
by a maximum, a minimum, and a crossover point at the λ max of the
absorption band.
The second derivative spectrum is characterized by two satellite
maxima and an inverted band of which the minimum corresponds to
the λ max of the fundamental band

A derivative spectrum therefore gives a better resolution of
overlapping bands than the corresponding fundamental spectrum and
may permit the accurate determination of the λ max of the individual
bands. Secondly, derivative spectrophotometry discriminates in
favor of substances of narrow spectral bandwidth against those with
broad bandwidth. The derivative amplitude (D), i.e. the distance
from a maximum to a minimum, is inversely proportional to the
fundamental spectral bandwidth (W) raised to the power (n) of the
derivative order. Thus,
D α (1 W )n
And consequently, substances with narrow spectral bandwidth
display larger derivative amplitudes than those with broad
bandwidth.
These advantages of enhanced resolution and bandwidth
discrimination found in derivative spectrophotometry permit the
selective determination of certain absorbing substances in samples in
which non-specific interference may limit the application of simple
spectrophotometric methods. Ephedrine hydrochloride in ephedrine
hydrochloride elixir is assayed by second derivative

spectrophotometry, which eliminates the broadband absorption of
the excipent (Beckett A. H and Stenlake J. B., 1997).
The enhanced resolution and bandwidth discrimination increases
with increasing order of derivative spectrophotometry. However, it
should be noted that the concomitant increase in electronic noise
inherent in the generation of the higher order spectra, and the
consequent reduction of the signal-to-noise ratio, would confer
practical limitations on the higher order spectra.
An important property of the derivative process is that broad bands
are suppressed relative to sharp ones. This effect increases with
increasing order of the derivative. This property leads to selective
rejection of broad, additive, spectral interferences such as Rayleigh
scattering (Moffat et. al, 1986).
-Instrumentation:
Derivative spectra can be obtained by any of three techniques. The
firstderivative spectra were obtained by modification of the optical
system. Spectrophotometers with dual monochromators set at small
wavelength intervals ( ∆λ , about 1-3 nm) apart or with the facility to
oscillate the wavelength over a small range, are required. In either
case the photo detector generates a signal, which has amplitude

proportional to the slope of the spectrum over the wavelength
interval.
The second technique employed to generate derivative spectra
comprise electronic differentiation of the spectrophotometer analog
signal. Resistance-capacitance (RC) modules may be incorporated
in the series between spectrophotometer and recorder to provide
differentiation of the absorbance, with respect to time and not
wavelength, thereby producing the signal d λ /dt. If the wavelength
scan rate is constant (d λ /dt = C), the derivative with respect to
wavelength is given by:
dA dλ = (dA/dt) (d λ /dt)= (dA/dt)(1/C)
Where:
dA dλ = Change of absorbance with respect to wavelength.
DA/DT = CHANGE OF ABSORBANCE WITH RESPECT TO
TIME.
d λ /dt = Change of wavelength with respect to time.
C = Concentration (mol/liter). Derivative spectra obtained with
RC modules are highly dependent on instrumental parameters, such
as the scan speed and the time constant. It is important therefore to
use a standard solution of the analyte to calibrate the measured value

under the instrumental conditions selected. The third technique is
based upon microcomputer differentiation. Microcomputers
incorporated into or interfaced with the spectrophotometer may be
programmed to provide derivative spectra during or after the scan of
the spectrum of the sample.
For quantitative purposes, the amplitude of a derivative peak can be
measured in a number of ways including peak- to -peak, peak-to-
satellite at short wavelength, peak-to-derivative zero, peak-to-
satellite at long wavelength and satellite peak-to- derivative zero.
If Beer-Lamber law is obeyed,
i.e.
A = ε bc
Then
dA dε
= . B.C
dλ dλ
d2A d 2ε
= . B.C
dλ2 dλ2
AND SO ON FOR A HIGHER DERIVATIVES.
Derivative spectrophotometry has found significant application in
clinical, forensic, and biomedical analysis (Moffat et. al, 1986 ).

1.2.1.2.8. CHEMICAL DERIVATISATION:
Derivatisations in direct spectrophotometric assays are based on the
conversion of the analyte by a chemical reagent to a derivative that
has different spectral characteristics.
When an excess of the reagent is used, in order to ensure complete
conversion, the absorbance of the derivative is usually proportional
to the concentration of the analyte. The majority of indirect
spectrophotometric procedures involve the conversion of the analyte
to a derivative, which will have a longer λ max and/or a higher
absorptiviy than the parent compound. Chemical derivatisation
procedures are adopted for any of several reasons:
1. IF THE ANALYTE ABSORBS WEAKLY IN THE
ULTRAVIOLET REGION
2. If the interference from irrelevant absorption is avoided by the
conversion of the analyte to a derivative, which absorbs in the
visible region, where irrelevant absorption is negligible.
3. To improve the selectivity of the assay of an ultraviolet-absorbing
substance in a sample that contains other ultraviolet-absorbing
components.
4. If the adoption of a visible spectophotometric procedure, instead
of an ultraviolet procedure is based on cost considerations.
1.2.2. CHROMATOGRAPHIC TECHNIQUES:
Chromatography was first discovered and named in 1906 by
Michael Tswett. It refers to separations of components of a sample
by distribution of the components between two phases: one, which is
stationary, and the other that moves, usually in a column (Gary and
James, 1986).
There are many types of chromatography. Size exclusion
chromatography is the one in which molecules are separated based on
their size by passage through a porous structure stationary phase. In ion
exchange and ion chromatography, ions are separated based on their
charge. In gas chromatography, gaseous substances are separated
according to their adsorption on or solubility in the stationary phase.
High performance liquid chromatography is a modern technique based
on the above principles but using micrometer size particles for the
stationary phase. Other types of chromatography include thin-layer
chromatography, paper chromatography, and electrophoresis in which
the stationary phase is in the form of a sheet or other flat surface and an
electrical gradient is applied across the sheet to cause molecules to
migrate according to the sign and magnitude of their charge.
All chromatographic techniques are principally based on establishment
of equilibrium between a stationary phase and a mobile phase. The
mechanism of retention however, differs for various type of
chromatography (Grayd Christian, 1998).
1.2.2.1. High Performance Liquid Chromatography
(HPLC):
Is a relatively modern analytical technique and it is preferred for the
rapid separation of non -volatile or thermally unstable samples. Its basic
mechanism is the same as liquid chromatography but it has the
advantage of high speed, good resolution, high sensitivity and its
convenience for quantitative analysis.
The difference between normal liquid chromatography (LC) and HPLC
is that for classical LC large porous particles are packed into columns
while in HPLC columns are packed with pellicular or micro particulate
packaging particles.
An increase in column efficiency with decrease porous particles
result from development of pellicular packing and micro particulate
which lead to use of short columns with smaller internal diameter.

The detector used in HPLC can either be selective e.g. it detects
ultraviolet absorbing or fluorescence compound or it can be
universal to detect all types of components.
The spectrophotometric and titration methods of assay are now
largely replaced by HPLC methods. This technique is preferred
because it allows the analysis of the drug faster and reasonably
without interference from excipent.
1.3. Analysis of chloroquine:
Choroquine phosphate tablets and injections are pharmacopeial
drugs available in both the British Pharmacopoeia and the United
State Pharmacopoeia. Chloroquine syrup is not a pharmacopeial
drug.
The method of analysis of tablets and injections in both pharmacopoeias
is non-aqueous titration. A high performance liquid chromatographic
(HPLC) method, which is not pharmacopeial, but a validated one is also
available (WHO, 1990).
A lot of work has been done concerning the analysis of chloroquine
phosphate. Of these, is work done by Nsimba SE,et,al, 2001. which
involved an HPLC analysis of sugar –coated chloroquine preparations
marketed in Tanzania versus an ordinary brand for an invivo and an in
vitro comparative study. Another HPLC method was done by Sjolin-

Forsberg G, et, al 1993. involving a long term follow up of chloroquine
phosphate concentration in skin suction fluid and plasma. The
concentration of chloroquine in plasma and whole blood was done by
an HPLC method in the work done by Neuvonen PJ, et al,1992. for the
study of prevention of chloroquine absorption by activated charcoal. A
bioavailability study of oral sugar-coated and plain formulation of
chloroquine phosphate marketed in Tanzania was done by Rimoy GH,
et, al, 2002, using an HPLC method. Allen LV Jr and Erichson MA,
1998, studied the stability of alprazolam, chloroquine phosphate,
cisapride, enalapril maleate and hydrazine hydrochloride in
extemporaneously compounded oral liquids by an HPLC method.
Hassan SS and Ahmed MA, 1991, did the determination of chloroquine
phosphate in various pharmaceutical preparations by polyvinyl chloride
(PVC) matrix membrane electrodes. Dwivedi AK, et, al, 2003, has
developed an HPLC and HPTLC method for assays of a new
antimalaria agent consisting of chloroquine phosphate, primaquine and
bulaquine. A conductmetric and indirect atomic absorption spectral
methods have been developed by Amin AS and Issa YM, 2003, for the
determinations of the antimalarials, amodiaquine hydrochloride,
chloroquine phosphate and primaquine phosphate. A first derivative
spectrophotometeric and HPLC determination of cinchocaine

hydrochloride in presence of it is acid degradation product was
developed by El-Gindy A, et, al, 1998.
In the presence work, a new derivative spectroscopic method has been
established for the analysis of chloroquine phosphate.
Objectives of the present work are:
2.1. Development and optimization of a derivative
spectrophotometric method for the analysis and quantitative
measurement of chloroquine phosphate.
2.2. Assessment of the selectivity, sensitivity and accuracy of the
developed method.
2.3. Employing the developed method for analysis of chloroquine
phosphate in pharmaceutical dosage forms.
2.4. Assessment of the selectivity of the method in presence of
pharmaceutical excipent.
2.5. Comparison between the developed method and the official
methods for analysis of chloroquine phosphate.
2.6. Application of the method in analysis of chloroquine
phosphate in biological fluids.

3.1. Instrumentation
3.1.1. A double-beam UV-VIS. Spectrophotometer, Unicam Heyios
α , model UVA 082017 with software and Epson printer/Germany,
was used in the quantitative analysis of different samples of
chloroquine phosphate.The spectral bandwidth was 2 nm and the
wavelength scanning speed was 1200 nm min-1.
The absorption spectra of test and reference solutions were recorded in
1- cm quartz cells over the range 200-400 nm. The zero order
absorption and the first derivative of the measured spectra were
obtained using accompanied software.
3.1.2. High Performance Liquid Chromatograph used was Syknm
connected to a UV/Visible dector S3200, Laboratory Computing

Integrator and S1121 pump /Japan. The separations were performed on
a Supelcosil LC-18, 15X4.6 mm at room temperature. The samples were
introduced through a bracket injector valve with 50 µl sample loop.
3.1.3. Memotitrator was used for non-aqueous titrations.
3.2. Reagents:
All reagents used were of analytical grade (perchloric acid, phosphoric
acid, sodium hydroxide, anhydrous acetic acid and chloroform).
Acetonitrile (HPLC grade) was used for preparation of mobile phase.
3.3. Formulations selected to be analyzed:
3.3.1. Amiquine tablets (Amipharma Laboratories, Ltd. Khartoum,
Sudan, batch no. t 0077, manufactured date Oct.2002, and expiry date
Oct.2005) labeled to contain 250 mg of chloroquine phosphate per
tablet, in addition to starch, talc, magnesium stearate and aerosol which
were used as exciepent.
3.3.2. Efroquine tablets (Efroze Chemical Industries (private) LTD.
Karachi Pakistan, batch no. B-42, manufactured date Jan/2000, and
expiry date Dec/2003) labeled to contain 250 mg of chloroquine
phosphate per tablet, in addition to acacia, aerosil, magnesium stearate,
starch, talc and titanium dioxide which were used as exciepent.

3.3.3. Lariago tablets (Ipcca Laboratories Ltd. India, batch
no.0013DQR, manufactured date 11/2000, and expiry date 10/2003)
labeled to contain 250 mg of chloroquine phosphate per tablet, in
addition to calcium stearate, starch, talc, acacia, gelatin, methyl paraben
and propyl paraben as exciepent.
3.3.4. Chloroquine phosphate tablets (Remidica Ltd. Cyprus-Europe,
batch no.16860, manufactured date 9/2001, and expiry date 9/2006)
labeled to contain 250 mg of chloroquine phosphate per tablet, in
addition to magnesium sterate, starch, talc, titanium dioxide, gelatin,
avicel and sucrose as exciepent.
3.3.5. Chloroquine phosphate injection (Bioluz Lab France, batch
no.22573, manufactured date 6/2002, and expiry date 6/2005) labeled to
contain 200 mg of chloroquine base per 5ml, in addition to phosphoric
acid.
3.3.6. Balsaquine injection (Balsam Pharmaceutical Company
Khartoum Sudan, manufactured date July/2001, and expiry date
July/2005) labeled to contain 200 mg of chloroquine base per 5 ml.
3.3.7. Chloroquine phosphate syrup (The Jordan Pharmaceutical
Manufacturing Co Ltd Jordan, batch no. 000839, manufactured date
8/2000, and expiry date 8/2003) labeled to contain 75 mg of

chloroquine base per 5ml, in addition to glycerin, propylene glycol,
sucrose, methyl paraben, propyl paraben, saccharin sodium and sodium
citrate as exciepent.
3.3.8. Lariago syrup (Ipcca Laboratories Ltd. India, batch no. DM
1001R, manufactured date Jun/2001, and expiry date Jun/2004) labeled
to contain 160 mg of chloroquine phosphate per 10 ml of syrup
equivalent to 100 mg of chloroquine base, in addition to raspberry
syrup, methyl paraben and propyl paraben as exciepent.
3.4. Stock Solutions of chloroquine standard:
3.4.1. Stock solutions of chloroquine phosphate standard:
An accurate weight (250 mg) of chloroquine phosphate standard was
transferred into a 250-ml volumetric flask, dissolved and diluted to
volume with water.
3.4.2. Stock solutions of excipent:
An accurate weight (Table 1) of each excipent was transferred into a
100-ml volumetric flask dissolved and diluted to volume with water
(Michael and Irene, 1995).

Table 1: Excipent used in chloroquine phosphate
formulations:
No. Name of excipent Weight/100ml
1 Aerosil 20 mg
2 Avicel 40 mg
3 Starch 48 mg
4 Magnesium stearate 82 mg
5 Lactose 24 mg
6 Talc 20 mg
7 Acacia 20 mg
8 Methyl paraben 20 mg
9 Propyl paraben 20 mg
10 Dicalcium phosphate 46 mg
11 Titanium dioxide 20 mg
12 Opadry 20 mg
13 Propylene glycol 500 mg
14 Glycerin 50 mg
15 Sucrose 1180 mg
16 Saccharin sodium 20 mg
17 Sodium citrate 50 mg
18 Citric acid 20 mg
19 Sorbitol 520 mg
20 Xanthan gum 20 mg
21 Tween 80 20 mg
22 Gelatin 20 mg
23 Phosphoric acid 455 mg
3.5. Solutions of pharmaceutical preparations (samples):
3.5.1. Stock solutions of chloroquine phosphate tablets:
Twenty tablets were accurately weighed, transferred to a mortar and
powdered. A mass containing 100 mg of chloroquine phosphate was
accurately weighed and transferred into a 100-ml volumetric flask,
dissolved in water, sonicated for 10 min and the volume completed to
100-ml with water. The solution was filtered through Whatman no. 40
filter paper.
3.5.2. Stock solutions of chloroquine phosphate injection:
2 ml of chloroquine phosphate injection was accurately measured using
a volumetric pipette and transferred to a 100-ml volumetric flask, and
the volume completed with water.

3.5.3. Stock solutions of chloroquine phosphate syrup:
A bottle of chloroquine phosphate syrup was transferred to a 100-ml
volumetric flask and the volume completed with water. 5 ml of the
solution was transferred to a 100 ml volumetric flask and the volume
completed to 100 with water.
3.6. Procedure:
3.6.1. Spectrophotometric method:
3.6.1.1. Calibration curve:
Serial dilutions were made from the stock standard solution to obtain
sets of concentrations of 2.5 µg ml-1, 10 µg ml-1, 20 µg ml-1, 30 µg ml-1,
40 µg ml-1, and 50 µg ml-1 with water.
Using water as blank, the zero order absorption and the first derivative
spectra of these solutions were recorded over the range 200-400 nm,
with a speed of 1200 nm/min, bandwidth 2 nm and medium smoothing
to the curve. The absorbance values at 221 nm (A221), 236 nm (A236), 256
nm (A256 ), 331 nm (A331), 343 nm (A343) were measured and the value of
first derivative at wavelengths 225 nm ( 1D225), 239 nm( 1D239), 260 nm (
1D ), 333 nm ( 1D333 ) and 349 nm ( 1D349 ) were measured (amplitude)

260
for each concentration. The regression, intercept and slope were
calculated for each wavelength.
The same procedure was followed for the construction of a calibration
curve for analysis of chloroquine phosphate standard dissolved in 0.01M
HCl.
3.6.1.2. Assay of chloroquine phosphate in presence of
excipent:
5 ml of the stock solution of each excipent were pipetted and
transferred to a 100 ml volumetric flask. To each, 5 ml of the stock
chloroquine phosphate standard solution were added and the volume
completed to 100 ml with water. 5 ml of the stock chloroquine
phosphate standard solution were diluted to 100 ml with water. Using
water as blank, the zero order absorption and the first derivative spectra
of these solutions were recorded over the range 200-400 nm, with a
speed of 1200 nm/min, bandwidth 2 nm and medium smoothing to the
curve. The absorbance values at ( A221), ( A236), ( A256), (A331), and ( A343)
were measured and the value of first derivative (1D225), (1D239), (1D260),
(1D333) and (1D349) were measured (amplitude) for each excipent and
standard solution. The concentration of chloroquine phosphate in each
sample was then calculated.
3.6.1.3. Assay of chloroquine phosphate in pharmaceutical
dosage form:
5 ml of the stock solution of each of chloroquine phosphate tablet,
injection and syrup were diluted to 100 ml with water. Using water as
blank the zero order absorption and the first derivative spectra of these
solutions were recorded over the range 200-400 nm, with a speed of
1200 nm/min, bandwidth 2 nm and medium smoothing to the curve.

The absorbance values (A221), (A236), (A256), (A331) and (A343) were
measured and the value of first derivative (1D225), (1D239), (1D260), (1D333)
and (1D349) were measured (amplitude) for each of the chloroquine
phosphate tablet solution, injection and syrup. The concentration in
each dosage form was calculated from regression equation for each
wavelength.
3.6.2. High Performance Liquid Chromatography Method:
The chromatographic procedure was carried out using, a mobile phase
consisting of a mixture of phosphate buffer (pH 3) and acetonitrile in
the ratio (3:2). The mobile phase was filtered and degassed before use
(Amal N. Abdelrahman et, al, 1994).
3.6.2.1. Phosphate buffer pH 3:
This was prepared by adding 7 ml of phosphoric acid (85%w/w) to
100 ml of water; 700 ml of water were then added, pH adjusted to 3
using 10 M NaOH and the volume completed to 1000 ml with water.
The separation was carried out using a C18 column with a flow rate of
1.5 ml/min, at wavelength 349 nm. The retention time was 3 min.
3.6.2.2. Calibration curve

From the stock standard solution, serial dilutions were made to obtain
sets of concentration of 2.5 µg ml-1, 10 µg ml-1, 20 µg ml-1, 30 µg ml-1,
40 µg ml-1, and 50 µg ml-1.
Triplicate injections (50 µl of each concentration) were made and the
average peak heights of the three readings were calculated. The
regression, slope and intercept of the curve were calculated.
3.6.2.3. Assay of chloroquine phosphate in presence of
excipent:
5 ml of the stock solution of each excipent were accurately measured
using a volumetric pipette and transferred to a 100 ml volumetric flask.
To each solution, 5 ml of the stock chloroquine phosphate standard
solution were added and the volume completed to 100 ml with water.
5 ml of the stock chloroquine phosphate standard solution was diluted
to 100 ml with water.
50 µl of the chloroquine phosphate standard solution were injected
(three times) then 50 µl of each excipent solution were injected (three
times).
After the injection of the third sample of each excipent solution,
50 µl of the standard chloroquine phosphate solution were again injected

(three times). The injection of the standard chloroquine phosphate
solution was repeated after each three samples solution (bracketing).
The average of the area under the curve (AUC) was calculated, and then
the concentration of chloroquine phosphate in each sample was
calculated.
3.6.2.4. Assay of chloroquine phosphate in pharmaceutical
dosage forms:
5 ml of the stock solution of each of chloroquine phosphate tablet,
injection and syrup were diluted to 100 ml with water. 50 µl of the
standard chloroquine phosphate solution were injected (three times)
followed by triplicate injection (50 µl each) of each sample solution.
After the injection of three samples, 50 µl of standard solution were
again injected (three times) and the injection of standard was repeated
after each three-sample solution (bracketing).
The average of area under the curve (AUC) was calculated and then the
concentration of chloroquine phosphate in each sample was worked out.
3.6.3. Non Aqueous Titration Method:
3.6.3.1. Assay of chloroquine phosphate tablets:

Twenty tablets were weighed and powdered. A mass containing 0.5 gm
of chloroquine phosphate was accurately weighed and transferred into a
separatory funnel, dissolved in 20 ml of 1.0 M sodium hydroxide and
extracted with 25 ml of chloroform (four times). Combined chloroform
extracts were evaporated to a volume of about 10 ml. 40 ml of
anhydrous acetic acid were added plus crystal violet (indicator). The
solution was titrated with 0.1M perchloric acid VS to color change of
indicator.
Each ml of 0.1M perchloric acid VS is equivalent to 25.79 mg of
chloroquine phosphate.
3.6.3.2. Assay of chloroquine phosphate injection:
A volume containing the equivalent of 0.4 gm of chloroquine base was
accurately measured and transferred into a separatory funnel, dissolved
in 20 ml of 1M sodium hydroxide and extracted with 25 ml of
chloroform (four times). Combined chloroform extracts were
evaporated to a volume of about 10 ml. 40 ml of anhydrous acetic acid
were added plus crystal violet (indicator). This was titrated with 0.1M
perchloric acid VS to color change of the indicator.
chloroquine base (BP).

3.6.3.3. Assay of chloroquine phosphate syrup:
(Non-official method, as BP tablet assay).
25 ml of chloroquine phosphate syrup were transferred to a separatory
funnel, dissolved in 20 ml of 1.0 M sodium hydroxide solution and
extracted with 25ml of chloroform (10 times). Combined chloroform
extracts were evaporated to a volume of about 10 ml. 40 ml of
anhydrous acetic acid was added plus crystal violet (indicator). This was
then titrated with 0.1M perchloric acid VS to color change of the
indicator.
3.7. Application of derivative spectroscopy in analysis of

chloroquine phosphate in biological fluids:
Trials for analysis of chloroquine phosphate in plasma by first derivative
spectroscopy were conducted. Following the validation of the method
of first derivative spectroscopy for analysis of chloroquine phosphate in
water, the method was applied for the analysis of chloroquine phosphate
dissolved in plasma and the results obtained were compared with the
absorbance spectroscopy results.
3.7.1. Calibration curve:
Prepared solutions of chloroquine phosphate standard in plasma in the
concentration range of (2.5 µ g ml-1 – 50 µ g ml-1) were assayed by first
derivative spectroscopy. From the results obtained calibration curves
were constructed.
3.7.2. Assay of chloroquine phosphate in pure plasma:
50 mg of chloroquine phosphate standard was accurately weighed and
dissolved in 50 ml of pure plasma (stock solution). 5 ml of this solution
were pipetted and transferred to a 100 ml volumetric flask and
completed to volume with water. 5 ml of pure plasma was diluted to 100
ml with water and used as a blank. 5 ml of the stock chloroquine
phosphate standard solution were diluted to 100 ml with water. The
zero order absorption and the first derivative spectra of these solutions
were recorded over the range 200-400 nm, with a speed of 1200
nm/min, bandwidth 2 nm and medium smoothing to the curve. The
absorbance values at 331 nm (A331) and 343 nm (A343) were measured
and the value of first order derivative at wavelengths 333 nm (1D333) and
349 nm (1D349) were measured (amplitude). No significant absorbance
bands were detected at wavelengths 221 nm, 236 nm and 256 nm for
zero order absorption and at 225 nm, 239 nm, and 260 nm for first
derivative. The concentration of chloroquine phosphate in each sample
was then calculated. The assay was repeated three times.

4.1. Calibration curves
Calibration curves for analysis of chloroquine phosphate standard by
different methods were done, these methods are:
Derivative spectrophotometry.
Absorbance spectrophotometry.
HPLC.
Standard stock solutions of chloroquine phosphate (1.0 mg ml-1)
were prepared by dissolving the compound in
(1) Water (2) 0.01M HCl (3) plasma

Standard solutions of chloroquine phosphate in the range 2.5-50 µg ml-
1
were prepared by the addition of the appropriate amount of the stock
standard solutions of the compounds to water or 0.01M HCl or pure
plasma. Measurements were made on three different days.
4.1.1. Derivative spectrophotometry:
A calibration curve was constructed by assaying standard solutions
of chloroquine phosphate in the range 2.5-50 µgml −1 in water, 0.01M
HCl and plasma.
The first derivative spectra of these solutions were recorded over the
range 200-400 nm against a blank of water, 0.01M HCl and plasma
respectively using UNICAM spectrophotometry.

The derivative value at 225 nm (1D225), 239 nm (1D239), 260 nm
(1D260), 333 nm (1D333) and 349 nm (1D349) were measured for
determination of chloroquine phosphate.
Table 2: Derivative spectrophotometry results for analysis

of chloroquine phosphate standard in H2O:
Concentration 1 1 1 1 1
-1
D225 D239 D260 D333 D349
µ g ml
50 -0.75 -0.34 -0.34 -0.15 -0.86

40 -0.65 -0.27 -0.27 -0.13 -0.68
30 -0.56 -0.20 -0.21 -0.09 -0.52
20 -0.37 -0.14 -0.14 -0.06 -0.35
10 -0.19 -0.07 -0.07 -0.03 -0.18
2.5 -0.04 -0.01 -0.02 -0.01 -0.04
Regression 0.988 0.999 0.999 0.997 1.000
Intercept 1.5*10-2 5*10-4 -2*10-3 -7*10-4 -4.1*10-3
Slope 1.5*10-2 -6.8*10-3 -6.8*10-3 -3.1*10-3 -1.71*10-2
Fig 1. First derivative spectrum of chloroquine phosphate
standard in water recorded over the range 200-400 nm
Fig.2. Calibration graphs for derivative spectrophotometry
analysis of chloroquine phosphate
standard in H2O at wavelengths 225 nm, 239 nm, 260 nm,
333 nm, 349 nm.
1 1
D 225 D239
0.0
-.2
-.4
D1
-.6
-.8
Observed
-1.0 Linear
0 10 20 30 40 50 60 Concentration
Concentration
ml- µ g
1
ml- µ g
1
Concentration
0.0
-.1
D1
-.2
-.3
Observed
-.4 Linear
0 10 20 30 40 50 60
Concentration
1 1
D260 D333
0.00
-.02
-.04
-.06
D1
-.08
-.10
-.12
-.14 Observe
-.16 Linear
0 10 20 30 40 50 60
Concentration Concentration
Concentration
ml- µ g ml- µ g
1 1
0.0
-.1
D1
-.2
-.3
Observe
-.4 Linear
0 10 20 30 40 50 60
Concentration
1
D349
0.0
-.2
-.4
D1
-.6
-.8
Observed
-1.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
µg ml -1
Table.3. Derivative spectrophotometry results for analysis of

chloroquine phosphate standard in 0.01M HCl:
Concentration 1 1 1 1 1
D225 D239 D260 D333 D349
µ g ml -1
50 -0.79 -0.34 -0.34 -0.15 -0.86

40 -0.67 -0.27 -0.27 -0.13 -0.7
30 -0.56 -0.21 -0.21 -0.09 -0.54
20 -0.37 -0.14 -0.14 -0.06 -0.36
10 -0.19 -0.07 -0.07 -0.03 -0.18
2.5 -0.04 -0.01 -0.01 -0.01 -0.04
Regression 0.987 0.999 0.999 0.995 0.999
Intercept -4.5*10-2 1.2*10-3 1.2*10-3 -7*10-4 -7.3*10-3
Slope 1.6*10-2 -6.9*10-3 -6.9*10-3 -3.1*10-3 -1.73*10-2
Fig. 3. Calibration graphs for derivative spectrophotometry
standard in 0.01M HCl at wavelengths (225nm, 239 nm, 260
nm, 333 nm, 349 nm)

1 1
D225 D239
0.0
-.1
D1
-.2
-.3
Observe
-.4 Linear
0 10 20 30 40 50 60
Concentration
ml- µ g ml- µ g
1 1
0.0
-.2
-.4
D1
-.6
-.8
Observe
-1.0 Linear
0 10 20 30 40 50 60
Concentration
1 1
D260 D333
0.0
-.1
D1
-.2
-.3
Observe
-.4 Linear
0 10 20 30 40 50 60
Concentration
ml- µ g ml- µ g
1 1
0.00
-.02
-.04
-.06
D1
-.08
-.10
-.12
-.14 Observed
-.16 Linear
0 10 20 30 40 50 60
Concentration
1
D349
0.0
-.2
-.4
D1
-.6
-.8
Observed
-1.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
ml-
1
µg
Table 4. Derivative spectrophotometry results for the
analysis of chloroquine phosphate standard in plasma
Concentration
1 1
µ g ml-1 D333 D349
50 -0.16 -0.7 8
40 -0.14 -0.63
30 -0.10 -0.47
20 -0.06 -0.32
10 -0.3 -0.16
2.5 -0.01 -0.05
Regression 0.993 1.000

Intercept 1.1*10-3 -9.2*10-3
Slope -3.3*10-3 -1.5*10-2
Fig. 4. Calibration graphs for derivative spectrophotometry
standard in plasma at wavelengths (333 nm, 349 nm)
1
D333
0.00
-.02
-.04
-.06
-.08
D1
-.10
-.12
-.14
-.16 Observed
-.18 Linear
0 10 20 30 Concentration
40 50 60
ml- µ g
1
Concentration
1
D349
0.0
-.2
D1
-.4
-.6
Observed
-.8 Linear
Concentration
0 10 20 30 40 50 -1 60
ml µ g
Concentration
4.1.2.Absorbance spectrophotometry:
of chloroquine phosphate in the range 2.5-50 µg ml-1 in water, in
0.01M HCl and plasma.
The zero order absorbance spectra of these solutions were recorded
over the range 200-400 nm against a blank of water, 0.01M HCl and
plasma respectively using UNICAM spectrophotometry.

The absorbance value at 221 nm A221, 236 nm A236, 256 nm A256,
331 nm A331 and 343 nm A349 were measured for determination of
Table 5: Absorbance spectrophotometry results for analysis

of chloroquine phosphate standard in water
Concentration
A221 A239 A256 A331 A343
µ g ml-1
50 2.65 1.72 1.5 1.58 1.66
40 2.18 1.34 1.15 1.25 1.32
30 1.71 1.01 0.87 0.95 1.01
20 1.15 0.67 0.58 0.63 0.67
10 0.57 0.33 0.28 0.32 0.34
2.5 0.13 0.06 0.06 0.07 0.08
Regression 0.997 1.000 1.000 1.000 1.000

Intercept 4.3*10-2 -2.29*10-2 -1.40*10-2 -5.90*10-3 3.16*10-2
Slope 5.33*10-2 3.45*10-2 2.93*10- 4.20*10-3 3.31*10-2
Fig 1. Zero absorbance spectrum of chloroquine phosphate

standard in water recorded over the range 200-400 nm
Fig.6. Calibration graphs for absorbance spectrophotometry
analysis of chloroquine phosphate standard in water at
wavelengths (221 nm, 236 nm, 256 nm, 2331 nm, 343 nm)
A221 A236
3.0
2.5
2.0
Absorbance
1.5
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
ml- µ g ml- µ g
1 1
2.0
1.5
Absorbance
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
A256 A333
1.6
1.4
1.2
1.0
Absorbance
.8
.6
.4
.2 Observe
0.0 Linear
0 10 20 30 40 50 60
Concentration
ml- µ g ml- µ g
1 1
1.6
1.4
1.2
1.0
Absorbance
.8
.6
.4
.2 Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
A343
2.0
1.5
Absorbance
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
ml- µ g
1
Table 6: Absorbance spectrophotometry results for analysis of
chloroquine phosphate standard in 0 .01M HCl
Concentration
A221 A239 A256 A331 A343
µ g ml-1
50 2.79 1.72 1.46 1.58 1.66

40 2.247 1.34 1.15 1.25 1.36
30 1.71 1.06 0.87 0.95 1.049
20 1.15 0.67 0.58 0.63 0.69
10 0.57 0.33 0.28 0.32 0.34
2.5 0.135 0.06 0.06 0.07 0.08
Regression 1.000 0.999 1.000 1.000 0.999
Intercept 1.1*10-2 -1.90*10-2 -1.22*10-2 -2.50*10-3 1.13*10-2
Slope 5.59*10-2 3.47*10-2 2.93*10- 6.16*10-2 3.35*10-2
Fig. 7.Calibration graphs for absorbance spectrophotometry
analysis of chloroquine phosphate standard in 0.01M HCl
at wavelengths (221 nm, 236 nm, 256 nm, 2331 nm, 343 nm)
A221 A236
3.0
2.5
2.0
Absorbance
1.5
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
ml- µ g
1
Concentration
ml- µ g
1
2.0
1.5
Absorbance
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
A256 A331
1.6
1.4
1.2
1.0
Absorbance
.8
.6
.4
.2 Observe
0.0 Linear
0 10 20 30 40 50 60
Concentration
ml- µ g ml- µ g
1 1
1.6
1.4
1.2
1.0
Absorbance
.8
.6
.4
.2 Observe
0.0 Linear
0 10 20 30 40 50 60
Concentration
A343
2.0
1.5
Absorbance
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
1
ml- µg
Table 7: Absorbance spectrophotometry results for analysis
of chloroquine phosphate standard in plasma
Concentration
A331 A343
µ g ml-1
50 2.21 2.19
40 1.82 1.77
30 1.41 1.38
20 1.08 1.06
10 0.52 0.51
2.5 0.23 0.23
Regression (r) 0.996 0.996
Intercept 0.1484 0.1438
Slope 4.18*10-2 4.12*10-2

Fig. 8.Calibration graphs for absorbance spectrophotometry
analysis of chloroquine phosphate standard in plasma at
wavelengths (331 nm, 343 nm)
A331
2.5
2.0
Absorbance
1.5
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
ml- µ g
1
A343
2.5
2.0
Absorbance
1.5
1.0
.5
Observed
0.0 Linear
0 10 20 30 40 50 60
Concentration
Concentration
ml-
1
µg
4.1.3.HPLC:
of chloroquine phosphate in the range 2.5-50 µg ml-1 in water. The
chromatograms of these solutions were recorded using SYKNM
instrument with UV/VS dectores at wavelength 349 nm and
acetonitrile: buffer pH 3 (3:2) as mobile phase with a flow rate at 1
ml min-1, retention time 3 min. The AUC was plotted against
concentration.
Table 14: High performance liquid chromatography results

for analysis of chloroquine phosphate standard
Concentration µ g ml-1 AUC
50 10.68
40 8.74
30 6.51
20 4.35
10 2.03
2.5 0.62
Regression (r) 0.999
Intercept 1.76*10-2
Slope 2.59*10-1
Fig .9. High performance liquid chromatography
chromatogram for analysis of cloroquine phosphate
standard in water
Time
Time
Time
Fig.10. Calibration graphs for high performance liquid
chromatography analysis of chloroquine phosphate
standard:
12
10
8
peak height
2
Observed
0 Concentration Linear
0 10 20 30 40 -1 µ g50 60
ml
Concentration
The HPLC method is more sensitive than both the zero absorption
and first derivative methods as both method can not detect
concentrations below 2.5 µ g ml-1 as the absorbance’s at these
concentration is almost zero while the HPLC method can detects ten
folds below 2.5 µ g ml-1

4.2. Analysis of Chloroquine Phosphate in presence of
pharmaceutical excipent:
There are about 23 different excipent used in chloroquine phosphate

formulations (tablet, syrup and injection) (Walter Lund, 1994).
The possibility of interference of these excipent on the analysis of
chloroquine phosphate was investigated. This was carried out by adding
each excipent, in the concentration used in the formulation, to a
standard chloroquine phosphate solution; the resultant mixture was
analyzed by three methods:
a) Absorbance spectrophotometry (zero order absorbance).
b) Derivative spectrophotometry (First order, proposed method).
c) HPLC (non-official validated method).
The concentration of chloroquine phosphate standard used was 1mg
ml-1 dissolved in distilled water. The weighed amount of excipent
(Table 1) was added to chloroquine phosphate standard solutions
and diluted to obtain a final concentration of 50 µgml −1 of the standard
chloroquine phosphate using distilled water.
The results of the first derivative spectrophotometry were compared
to that of absorbance spectrophotometry (the zero order absorption)
at different wavelengths of 221nm, 236 nm, 256 nm, 331 nm and

343 nm. It was also compared to the results of HPLC. The mean of
the results of each method in the three different days were plotted
against wavelengths.
The precision and accuracy of the first derivative was compared to
HPLC and zero order absorbance results, using F-test and t-test.
The F-test and t-test results were compared to tabulated values
(Grayd Christian, 1998), which are:
The theoretical F at 95% confidence level and degree of freedom Ø1
=2
Ø2 = 2 for first derivative method, zero order absorbance method
and HPLC method were found to be 19.0 (theoretical).
(If experimental F did not exceed theoretical value at selected
confidence level, then there is no significant difference between the
two methods and the proposed method is precise)
The theoretical t at the 95% confidence level and degree of freedom
Ø1 = 2
Ø2 = 2 for first derivative method, zero order absorbance method
and HPLC method were found to be 4.303 (theoretical).

(If experimental t did not exceed theoretical value at selected
confidence level, then there is no significant difference between the
two methods and the proposed method is accurate)
4.2. Analysis of Chloroquine Phosphate in presence of

pharmaceutical excipent:
There are about 23 different excipent used in chloroquine phosphate formulations

(tablet, syrup and injection) (Walter Lund, 1994) (15),
The possibility of interference of these excipent on the analysis of
chloroquine phosphate was investigated. This was carried out by adding each
excipent in the concentration used in the formulation to a standard
chloroquine phosphate solution, the resultant mixture was analyzed by three
methods:
a) Absorbance spectrophotometry (zero order absorbance).
b) Derivative spectrophotometry (First order, proposed method).
c) HPLC (non-official validated method).
The concentration of chloroquine phosphate standard used was 1mg ml-1
dissolved in distilled water. The weighed amount of excipent (Table 1)
was added to chloroquine phosphate standard solutions and diluted to
obtain a final concentration of 50 µgml −1 of the standard chloroquine
phosphate using distilled water.
The results of the first derivative spectrophotometry were compared to
that of absorbance spectrophotometry (the zero order absorption) at

different wavelengths of 221nm, 236 nm, 256 nm, 331 nm and 343 nm. It
was also compared to the results of HPLC. The mean of the results of
each method in the three different days were plotted against wavelengths.
The precision and accuracy of the first derivative was compared to HPLC
and zero order absorbance results, using F-test and t-test.
The F-test and t-test results were compared to tabulated (Grayd Christian)
(16), which are:
F at 95% confidence level and degree of freedom 2 for first derivative
method, zero order absorbance method and HPLC method was found to
be 19.0
(If calculated F did not exceed tabulated value at selected confidence
level then there is no significant difference between the two methods and
the proposed method is precise)
t at the 95% confidence level and degree of freedom 2 for first derivative
method, zero order absorbance method and HPLC method was found to
be 4.303
(If calculated t did not exceed tabulated value at selected confidence level
then there is no significant difference between the two methods and the
proposed method is accurate)

4.3. Excipent used in Chloroquine Phosphate formulations:
There are many excipent used in Chloroquine Phosphate formulations
(Table1). The effect of these excipent on the analysis of chloroquine
phosphate was studied using:
a) Absorbance spectrophotometry.
b) Derivative spectrophotometry.
c) HPLC.
The excipent were classified according to the pharmaceutical uses
(Michael and Irene Ash,1995) (20), the effect of each group on the
analysis of chloroquine phosphate by the different methods was studied
and the results were compared.
1. Disintegrants:
The disintegrants used in chloroquine phosphate formulations are:

1) Starch 2) Aerosil 3) Avicel
Fig.11. Results of analysis of chloroquine phosphate
in presence of starch
Absorbance
105 Derivative
chloroquine phosphate
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of aerosil
Absorbance
105
Derivative
HPLC
percentage w/w of
103
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of avicel
105 Absorbance
Derivative
103
percentage w/w 0f
HPLC
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of magnesium stearate
105 Absorbance
Derivative
103 HPLC
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of lactose
Absorbance
Derivative
105
HPLC
percentage w/w of
103
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of talc
Absorbance
105 Derivative
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of accacia
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of meyl paraben
Absorbance
180 Derivative
160 HPLC
percentage w/w of
140
120
100
80
60
40
20
0
225nm 239nm 260nm 333nm 349nm
wave length
in presence of propyl paraben
Absorbance
Derivative
160
HPLC
140
percentage w/w of
120
100
80
60
40
20
0
225nm 239nm 260nm 333nm 349nm
wave length
in presence of dicalcium phosphate
Absorbance
Derivative
105 HPLC
percentage w/w of
103
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave l ength
in presence of titanium dioxide
Absorbance
Derivative
HPLC
105
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of opadry
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of propylene glycol
Absorbance
Derivative
105
HPLC
103
phosphate
percentage w/w of
chloroquine
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of glycerin
Absorbance
Derivative
105 HPLC
percentage w/w of
103
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of sucrose
Absorbance
Derivative
105
HPLC
103
phosphate
chloroquine
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of saccharin sodium
Absorbance
Derivative
250
HPLC
200
percentage w/w of
150
100
50
0
225nm 239nm 260nm 333nm 349nm
wave length
in presence of sodium citrate
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of citric acid
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of sorbitol
Absorbance
105 Derivative
HPLC
ofchloroquine phosphate
percentagew/w
103
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of xanthan gum
Absorbance
Derivative
105
HPLC
103
chloroquine
percentage w/w of
h t
101
h
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of tween 80
Absorbance
105 Derivative
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of gelatin
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
in presence of phosphoric acid
Absorbance
Derivative
105
HPLC
103
percentage w/w of
101
99
97
95
225nm 239nm 260nm 333nm 349nm
wave length
4.3.1. Disintegrants:
The disntegrants used in chloroquine phosphate formulations are:
1) Starch 2) aerosol 3) avicel
Fig.34. Comparison of the results of analysis of chloroquine phosphate
in presence of disintegrants
a. Absorbance b. Derivative c. HPLC
starch starch starch
aerosil aerosil
aerosil
avicel avicel
choroquine phosphate
avicel
percentage w/w of
105 105 105
percentage w/w of
103 percentage w/w of
103 103
101 101
101
99 99
99
97 97
97
95 95
95
m
m
1n
6n
6n
1n
3n
5n
9n
0n
3n
9n starch aerosil avicel

22
23
25
33
34
22
23
26
33
34
wave length wave length

4.3.2. Lubricant:
The lubricants used in chloroquine phosphate formulations are:
1) Mg Stearate 2) Talc
in presence of lubricant
Mg Sterate
Mg Sterate Mg Sterate
Talc
Talc Talc
105 105
105
percentage w/w of
percentage w/w of
percentage w/w of
103 103
103
101 101 101
99 99 99
97
97 97
95
95 95
m
Mg Sterate Talc
1n
6n
6n
1n
3n
m
5n
9n
0n
3n
9n
22
23
25
33
34
22
23
26
33
wave length wave length34

4.3.3. Diluents:
The diluents used chloroquine phosphate formulations are:
1) Lactose 2) Dicalcium phosphate 3) Sucrose
in presence of diluents
Lactose Lactose Lactose
DiCal Phos. DiCal Phos.

DiCal Phos.
Sucrose
Sucrose Sucrose 105
105 105
percentage w/w of
percentage w/w of
103
percentage w/w of
103 103
101
101 101
99
99 99
97 97
97
95 95
95
e
e
os
os
os
m
Ph
1n
6n
6n
1n
3n
cr
ct
5n
9n
0n
3n
9n
La
Su
22
23
25
33
34
22
23
26
33
34
al
wavelength
iC
wave length
D
4.3.4. Coloring Agent:
The coloring agents used in chloroquine phosphate formulations are:
1) Titanium dioxide 2) Opadry
Fig37. Comparison of the results of analysis of chloroquine phosphate
in presence of coloring agents
Titnium dioxide Titnium dioxide Titnium dioxide
Opadry Opadry Opadry
105
105 105
percentage w/w of
103
percentage w/w of
103
percentage w/w of
103
101 101
101
99 99
99
97 97 97
95 95 95
Titnium Opadry
m
dioxide
m
m
1n
6n
6n
1n
3n
5n
9n
0n
3n
9n
22
23
25
33
34
wave length
22
23
26
33
34
wave length
4.3.5. pH Adjustment excipent:
The pH adjustment excipent used in chloroquine phosphate formulations are:
1) Sodium citrate 2) citric acid 3) phosphoric acid
Fig.38 Comparison of the results of analysis of chloroquine phosphate
in presence of pH adjustment agents by the three methods
Sodium Citrate Sodium Citrate Sodium Citrate
Citric Acid Citric Acid

Citric Acid
Phosphoric Acid Phosphoric Acid
105 Phosphoric Acid 105
105
percentage w/w of
phosphate
chloroquine
percentage w/w of
103 103
phosphate
chloroquine
percentage w/w of
103
101 101
101
99 99
99
97 97
97
95 95
95
Citric Acid
Phosphoric
Sodium
Citrate
m
nm
m
m
1n
6n
6n
1n
3n
5n
0n
3n
9n
Acid
29
22
23
25
33
34
22
26
33
34
wave length wave length
4.3.6. Anti-microbial agents:

The Anti-microbial excipent used in chloroquine phosphate formulations are:
1) Methyl paraben 2) propyl paraben 3) propylene glycol
4) Saccharin sodium 5) saccharin sodium
in presence of anti-microbial agents
methyl paraben methyl paraben methyl paraben
propyl paraben propyl paraben propyl paraben
propylene glcol propylene glcol propylene glcol
glycerin glycerin glycerin
250 saccharin sodium saccharin sodium 105 saccharin sodium
180
percentage w/w of
percentage w/w of
160 103
phosphate
percentage w/w of
chloroquine
200
140
120 101
150
100
99
100 80
60 97
50 40
20 95
0 0
r in
pa n
en en
um
co
e
ab
ha yce
gl
di
ra
so
pr par
e
m
gl
m
m
1n
6n
6n
1n
3n
rin
5n
9n
0n
3n
9n
yl
yl
hy
22
23
25
33
34
op
op
22
23
26
33
34
et
cc
wave length
pr
wave length
sa
4.3.7. Emulsifying agent:
The emulsifying excipent used in chloroquine phosphate formulations are:
1) Acacia 2) Gelatin 3) Sorbitol 4) Xanthan Gum 5) Tween 80
in presence of emulsifing agents by the three methods
accacia Accacia Accacia
gelatin Gelatin Gelatin
sorbitol Sorbitol Sorbitol
xanthan gum Xanthan gum Xanthan gum
tween 80 Tween 80 Tween 80
105 105 105
percentage w/w of
percentage w/w of
103
percentage w/w of
103 103
101 101 101
99 99
99
97 97
97
95 95
95
m
ia
80
l
in
ito
m
at
c
gu
ca
rb
n
1n
6n
6n
1n
3n
el
m
ee
So
n
Ac
G
5n
9n
0n
3n
9n
22
23
25
33
34
ha
Tw
22
23
26
33
34
nt
wave length
Xa
wave length
Fig.41. Results of analysis of chloroquine phosphate injection
Absorbance
Derivative
HPLC
105
Non Aqueous Titration
103
percentage w/w of
101
99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.42. Results of analysis of Balsaquine injection
Absorbance
Derivative
HPLC
Non AqueousTitration
105
percentage w/w of
103
101 Non AqueousTitration
99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.43. Results of analysis of Efroquine tablets
Absorbance
Derivative
HPLC
105
phosphate
percentage w/w of chloroquine
103
101

99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.44. Results of analysis of chloroquine phosphate tablets
Absorbance
Derivative
HPLC
107
105
percentage w/w of
103
101
99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.45. Results of analysis of Amiquine tablets by the four methods:
Absorbance
Derivative
HPLC
105
103
percentage w/w of

101
99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.46. Results of analysis of Lariago tablet by the four methods:
Absorbance
Derivative
HPLC
NonAqueousTitration
107
105
percentage w/w of
103
101
NonAqueousTitration
99
97
95
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.47. Results of analysis of chloroquine syrup (JPM)
Absorbance
Derivative
HPLC
250
200
percentage w/w of
150

100
50
0
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.48. Results of analysis of Lariago syrup
Absorbance
Derivative
HPLC
180
160
percentage w/w of
140
120
100
80
60
40
20
0
225 nm 239 nm 260 nm 333 nm 349 nm
wave length
Fig.49. Results of analysis of chloroquine phosphate in pure plasma by
the first derivative and zero order absorption spectroscopy
absorption
derivative
160
140
percentage w/w of
120
100
80
60
40
20
0
333 nm 349 nm
wave length
4.4. Analysis of chloroquine phosphate in pharmaceutical dosage
forms:
Samples of different dosage forms of chloroquine phosphate locally manufactured and imported that covered the 23 excipent used in
chloroquine phosphate formulation analyzed by:-
1) Absorbance spectrophotometry (Zero order absorbance)
2) Derivative spectrophotometry (First order, proposed method)
3) HPLC (non-official validated method)
4) Non -Aqueous Titration (official method, BP)
The pharmaceutical formulations used included both branded and generic
products. Four tablet dosage forms were used (Amiquine tablets,
Efroquine tablets, Lariago tablets and Chloroquine Phosphate tablets).
Two syrup dosage form (Lariago syrup and Chloroquine Phosphate
syrup) and two injectable (Balsaquine injections and Chloroquine
Phosphate injections) were used. The results of the analysis of
chloroquine phosphate in the different brands of the different dosage
forms by first derivative spectrophotometer compared with results
obtained by the other three methods. F and t-tests used to compare
precision and accuracy of first derivative spectrophotometry method to
the other three methods.

5.1. Discussion
The aim of the present work is to propose a new analytical method for the
assay of chloroquine phosphate in pharmaceutical dosage forms such as
tablet, syrup and injection. The official method for the assay of
chloroquine phosphate tablets and chloroquine phosphate injections (BP
and USP) is non-aqueous titration. This titremetric method is a sensitive
one. However, since it depends on the extraction of chloroquine,
phosphate from it is formulation; there may be some variation in
extraction from one analyst to another. Moreover, chloroform is rather
expensive. In addition, the method is not suitable for analysis of
chloroquine syrup as its extraction is difficult (due to emulsion
formation). There is also the need for large amounts of chloroform and
long period to extract the drug from its formulation. Hence, the sensitivity
of this method is questioned.
Another method for analysis of chloroquine phosphate dosage form is an
HPLC method, which is non –official (WHO method). It is a validated
method for analysis of all chloroquine phosphate dosage forms. It is a
sensitive method but it is time consuming and expensive.
As chloroquine is an important drug of high use and consumption in
Sudan its analysis has attracted great attention.

In the present work, a spectrophotometric method of assay of chloroquine
was developed and optimized.
The results of analysis of chloroquine in different pharmaceutical dosage
forms obtained by this method were compared to the results obtained by
the official titremetric BP method and the HPLC method (W H O).
Comparisons were made with regard to developing and adopting the new
method based on speed, simplicity, sensitivity, selectivity, accuracy,
precession, reproducibility, cost and ability to employ in routine analysis
of the drug.
5.1. 1. Derivative spectrophotometry calibration curves:
Calibration curves were constructed from the results of assaying standard
solutions of chloroquine phosphate in water and in 0.01M HCl.
The first derivative spectra of these solutions were recorded over the range
200- 400 nm (fig.1).
Under the described experimental conditions, the calibration curves
obtained by plotting 1D values versus concentrations at the wavelengths
225 nm (1D225), 239 nm (1D239), 260 nm (1D260), 333 nm (1D333) and 349
nm (1D349), showed linear relationships in the concentration range 2.5-
50 µg ml-1.
The calibration curves were in agreement with the Beers law.

Comparison was made between the results obtained using water and 0.01
M HCl as solvents.
Table 9: Statistical analysis of the calibration graphs of chloroquine
phosphate using first-derivative spectrophotometry:
Wave length Solvent Intercept Slope Regression

(nm) coefficients (r)
1.5*10-2 1.5*10-2 0.988
225 Water
0.01M HCl -4.5*10-2 1.6*10-2 0.987
5*10-4 -6.8*10-3 0.999
239 Water
0.01M HCl 1.2*10-3 -6.9*10-3 0.999
-2*10-3 -6.8*10-3 0.999
260 Water
0.01M HCl 1.2*10-3 -6.9*10-3 0.999
-7*10-4 -3.1*10-3 0.997
333 Water
0.01M HCl -7*10-4 -3.1*10-3 0.995
-4.1*10-3 -1.71*10-2 1.000
349 Water
0.01M HCl -7.3*10-3 -1.73*10-2 0.999
From the above results, it is evident that there is no significant difference
in linearity of chloroquine calibration curve in water and in 0.01M HCl. It
was therefore decided to use water as the solvent in the analysis carried
out in this work.

The chloroquine calibration curves were linear in all selected
wavelengths. However, the best linearity was observed at wavelength 349
nm.
5.1.2. Absorbance spectrophotometry calibration curves:
Calibration curves were constructed from the results obtained by assaying
standard solutions of chloroquine phosphate in the range 2.5-50 µg ml-1 in
water and in 0.01M HCl.
The zero order absorbance spectra of these solutions were recorded over
the range 200-400 nm (fig. 5).
Under these experimental conditions the calibration curves, obtained by
plotting absorbencies values versus concentration at the wavelengths 221
nm (A221), 236 nm (A236), 256 nm (A256), 331 nm (A331) and 343 nm
(A343), showed linear relationships in the concentration range 2.5-50

-1
µg ml
The calibration curves were in agreement with the Beers law.
The results obtained using water and 0.01M HCl as solvents were
compared.
phosphate using zero-order absorbance spectrophotometry:
Wavelength Solvent Intercept Slope Regression

(nm) coefficients (r)
5.33*10-2 4.3*10-2 0.997
221 Water
0.01M HCl 1.1*10-2 5.59*10-2 1.000
-2.29*10-2 3.45*10-2 1.000
236 Water
0.01M HCl -1.9 *10-2 3.47*10-2 0.999
-1.40*10-2 2.93*10-2 1.000
256 Water
0.01M HCl -1.22*10-2 2.93*10-2 1.000
-5.90*10-3 3.16*10-2 1.000
331 Water
0.01M HCl -2.50*10-3 3.16*10-2 1.000
4.20*10-3 3.31*10-2 1.000
346 Water
0.01M HCl 1.13*10-2 3.35*10-2 0.999
It is seen from the above results that there is no significant difference in
the result when either water or 0.01M HCl were used.
It was therefore decided to use water as the solvent.
phosphate using first derivative, zero-order absorbance
spectrophotometry and HPLC methods:
Regression
Method Wavelength Intercept Slope
coefficients (r)
5.33*10-2 4.3*10-2 0.997
Absorbance 221nm
1.5*10-2 1.5*10-2 0.988
Derivative 225nm
-2.29*10-2 3.45*10-2 1.000
Absorbance 236nm
5*10-4 -6.8*10-3 0.999
Derivative 239nm
-1.40*10-2 2.93*10-2 1.000
Absorbance 256nm
-2*10-3 -6.8*10-3 0.999
Derivative 260nm
-5.90*10-3 3.16*10-2 1.000
Absorbance 331nm
-7*10-4 -3.1*10-3 0.997
Derivative 333nm
4.20*10-3 3.31*10-2 1.000
Absorbance 346nm
-4.1*10-3 -1.71*10-2 1.000
Derivative 349nm
1.76*10-2 2.59*10-1 0.999
HPLC 349nm
It can be seen that linear relationships were obtained in the concentration
range of 2.5-50 µg ml-1 for all three analytical methods.
The linearity of the calibration graphs and the adherence of the systems to
Beers law were validated by the values of correlation coefficient of the
regression equation, which were 0.99 to 1.000 for all methods.
The values of intercept were close to zero in all methods.
5.1.3. Analysis of chloroquine phosphate in presence of excipent:
Twenty-three different excipent known to be used in chloroquine
phosphate formulations (tablets, syrup and injection) (Table 1) were used.
The possible interference of these excipent with the result of analysis of
chloroquine phosphate was investigated. This done by adding each excipent,

in the concentration used in the formulation, to a standard solution of
chloroquine phosphate, which was then analyzed by the three analytical
methods
a) Absorbance spectrophotometry (Zero Absorbance)
b) Derivative spectrophotometry (First order; proposed method)
c) HPLC (non-official validated method)
The analysis was carried out by pipetting 5 ml of stock solution of each
excipent into a 100 ml volumetric flask. To each of these, 5 ml of stock
standard chloroquine phosphate solution were added and the volume
completed to 100 ml with water to obtain a final concentration of 50 µg ml-1
of chloroquine phosphate standard. Each solution was then analyzed by the
three different methods and the analysis was carried out three times in three
different days.
The zero order absorption and the first derivative spectra of these solutions
were recorded over the range 200-400 nm, with a speed of 1200 nm/min,
bandwidth 2 nm and medium smoothing to the curve. The HPLC method
was carried out using a mobile phase of a mixture of phosphate buffer pH3 :
acetonitrile (3:2).
The was separation carried out using a C18 column with a flow rate of 1.5
ml/min, at wavelength 349 nm. The retention time was 3 min (fig.9).
The results obtained by the first derivative spectrophotometry were
compared to those obtained by the absorbance spectrophotometry at the
selected wavelengths and compared to the result obtained by the HPLC
(as in tables).
The precision and accuracy of derivative method were compared to those
of HPLC and Zero order absorbance by calculating F- and t, which has
been compared to theoretical ones.
The theoretical F at the 95% confidence level and degree of freedom Ø1 =
2 Ø2 =2
(19.0)
While the theoretical t at the 95% confidence level and degree of freedom
Ø1 = 2
Ø2 =2 (4.303).
analysis of chloroquine phosphate standard solutions each containing,
separately, each of the excipent (starch, magnesium sterate, lactose, talc,
acacia, dicalcium phosphate, titanium dioxide, Opadry, propylene glycol,
glycerin, sucrose, sodium citrate, citric acid, sorbitol, xanthan gum, tween
80, gelatin, phosphoric acid) by the three methods showed no variation in
the results. This reveals that there was no interference with the analysis of
chloroquine phosphate resulting from any of these exciepent. There were
no significant differences between the results obtained by first derivative

spectrophotometry method, HPLC and zero order absorption methods (as
shown in tables).
Experimental F and t values were smaller than the theoretical theoretical
value.
The first derivative spectrophotometry method was thus found to be
precise and accurate, being more so at wavelength 349 nm compared to
zero absorption and HPLC methods. The results were almost identical at
this wavelength.
The proposed derivative spectrophotometry method thus proved suitable
for assay of the drug in presence of pharmaceutical exciepent.
Result of analysis of chloroquine phosphate standard solutions containing
aerosil showed a significant difference at wavelength 225 nm, between
derivative and HPLC methods and at 260 nm between derivative and
absorbance methods (Table17). It is concluded that derivative
spectroscopic analysis of chloroquine phosphate was not accurate and
precise at these wavelength for the analysis of chloroquine phosphate
containing aerosil. This may be due to low selectivity at small
wavelength.
The results of analysis of standard chloroquine phosphate solution
containing avicel showed significant differences at wavelength 239 nm,
between derivative and HPLC methods (Table17). The derivative method
is therefore not recommended at this wavelength for the analysis of

chloroquine phosphate containing avicel as selectivity decreased with
decreased wavelength.
Analysis of standard chloroquine phosphate solution containing methyl
paraben or propyl paraben, or saccharin sodium showed that there were
significant differences at all wavelengths except 333 nm and 349 nm,
between the first derivative and HPLC methods, the first derivative, and
zero order absorption methods (see remark on tables 29, 31 and
44respectively).
It can be concluded from these results that the first derivative and zero
order absorption methods were not suitable at these wavelengths with
regard to analysis of chloroquine phosphate containing methyl paraben,
propyl paraben or saccharin sodium, while the HPLC method is very
suitable and superior to the two spectrophotometric methods as it is
highly selective.
Methyl paraben, propyl paraben and saccharin sodium appear to interfere
in the analysis of chloroquine phosphate at all wavelengths except at
wavelength 333 nm and 349 nm in the zero order absorption method and
the first derivative method (fig.50).
This is understandable since one would except higher selectivity with
increased wavelength of absorption and therefore absence of interference
of these exciepent at 333 nm and 349 nm.

Fig. 50. Zero order absorbance and first derivative spectra for
analysis of chloroquine phosphate standard solutions in presence of
saccharin sodium
5.1.4. Analysis of chloroquine phosphate in pharmaceutical dosage forms:
Samples of different brands of different dosage forms of chloroquine
phosphate both locally manufactured and imported that covered the 23
excipent used in chloroquine phosphate formulations were analyzed by the
three methods mentioned above in addition to the official non -aqueous
titration method.
The samples included both branded and generic products.
These were: (A) four-tablet dosage forms; Amiquine tablets, (Amipharma
Laboratories, Ltd. Khartoum, Sudan), Efroquine tablet (Efroze Chemical
Industries (private) LTD. Karachi, Pakistan), Lariago tablet (Ipca Laboratories
Ltd, India) and chloroquine phosphate tablet (Remidica Ltd. Cyprus-Europe).

(B) two syrup dosage forms; Lariago syrup (Efroze Chemical Industries
(private) LTD. Karachi, Pakistan) and Chloroquine phosphate syrup (The
Jordan Pharmaceutical Manufacturing Co Ltd, Jordan).
(C) Two injectables, Balsaquine injection (Balsam Pharmaceutical Ltd,
Khartoum, Sudan) and Chloroquine phosphate injection (Bioluz Lab,
France).
The analysis of chloroquine phosphate in dosage forms was conducted by
diluting 5 ml of stock solution of chloroquine phosphate from tablets,
injections and syrups to 100 ml with water. Using water as blank, the zero
order absorption and the first derivative spectra of these solutions were
recorded over the range 200-400 nm, with speed 1200 nm/min,
bandwidth 2 nm and medium smoothing to the curve. The HPLC method
was carried out using a mobile phase of a mixture of phosphate buffer
(pH 3): acetonitrile (3:2).
The separation was carried out using a C18 column with a flow rate of 1.5
ml/min, at a wavelength of 349 nm; the retention time was 3 min (fig.9).
The non-aqueous titration method carried out after extraction of the
samples with chloroform and evaporation of the chloroform to dryness.
The residue was dissolved in anhydrous acetic acid and then titrated with
0.1M perchloric acid using crystal violet as indicator.

The results of assay of the different dosage forms of chloroquine
phosphate by first derivative spectrophotometry were compared with the
results obtained from the three other methods. F and t-tests were used to
compare precision and accuracy of first derivative spectrophotometry
method with the other three methods.
The experimental F-value and t-value were compared to the theoretical
ones.
The results of assay of chloroquine phosphate injection dosage forms
showed no significant differences when carried out by either of the first
derivative spectrophotometry, HPLC, zero order absorption
spectrophotometry and non-aqueous titration methods.
The experimental F and t values were smaller than the theoretical ones
(see tables 68 and 70). It was therefore concluded that first derivative
spectrophotometry was precise and accurate at all wavelengths; result
being almost identical (as F-value and t-value were almost zero).
Similar results were obtained in the analysis of chloroquine phosphate in
tablet dosage forms. There were no significant differences between first
derivative spectrophotometry, HPLC, zero order absorption and non-
aqueous titration methods of analysis.
The experimental F and t values were smaller than the theoretical ones
(see tables 72, 74, 76 and 77). The first derivative spectrophotometry
was thus considered precise and accurate at all wavelengths. It was noted
that this is particularly so at wavelength 349 nm as results were almost
identical (F-value and t-value were almost zero).
Statistical analysis of the results obtained for chloroquine phosphate in
syrup dosage forms showed that there were significant differences
between first derivative spectrophotometry, zero order absorption and
non-aqueous titration methods of assay at all wavelengths except at
wavelength 333 nm and 349 nm (see tables 79and 81). The experimental
F and t values were greater than the theoretical ones.
However, comparison of the results obtained by the first derivative
method and the HPLC method revealed no significant differences when
analysis was conducted at wavelength 260 nm, 333 nm, and 349 nm for
chloroquine phosphate syrup.
It is evident from the results that the first derivative spectrophotometry is
precise and accurate at these wavelengths. It is suggested that the first
derivative method of analysis could be used for assay of chloroquine
phosphate in syrup dosage form using wavelength 260 nm, 333 nm or
349 nm.
5.1.5. Application of the spectrophotometric derivative method in the
in vitro analysis of chloroquine phosphate in biological fluids:
The first derivative spectrophotometric method was employed to analyze
chloroquine phosphate in biological fluids (pure plasma).100 mg of
chloroquine phosphate were dissolved in 100 ml of plasma and serial

dilutions made by distilled water to obtain a set of concentration in the
range of 2.5-50 µ g ml-1. From the results of analysis, a calibration curves
was constructed.
The first order derivative spectra of these solutions were recorded over
the range 200-400 nm against blank plasma diluted with water.
The derivative values at 333 nm (1D333), 349 nm and (1D349) were
measured for determination of chloroquine phosphate.
Linear relationships were obtained in the concentration range 2.5-
50 µg ml-1. On the other hand, determination of chloroquine phosphate, in
plasma using zero order absorption values at 331 nm (A333) and 343 nm
(A343), was not feasible.
This is attributed to possible interference from plasma components. The
zero order absorption method clearly lacks selectivity in biological fluids.
Application of first order derivative spectrophotometry was found to
correct for plasma interference and to enhance selectivity. By measuring
the values of the 1D amplitudes at 333 nm and 349 nm, the concentration
can directly be calculated since the first derivative measurement cancels
the irrelevant absorbance due to plasma components at these
wavelengths.
phosphate in plasma using first-derivative spectrophotometry:

Method Wavelength Intercept Slope Regression (r)
1.1*10-3 -3.3*10-3 0.993
Derivative 333nm
-9.2*10-3 -1.5*10-2 1.000
Derivative 349nm
Fig. 51. Zero order absorbance and first derivative spectra for
analysis of chloroquine phosphate standard solutions in plasma

Derivative spectroscopy has been widely applied in the analysis of
different pharmaceutical dosage forms. It solves the problems of analysis
associated with drug combinations, stability studies of drugs and
degradation products, drug impurities, interference of excipent in drugs,
topical preparations analysis like creams and ointments and it also solves
the problem of analysis of drugs in biological fluids.
A lot of work was reported for the application of derivative methods for
solving these problems. Nevin Erk, 2001, reported the analysis of binary
mixtures of lostran potassium and hydrochlorothiazide. The assay of
ephedrine hydrochloride and theophylline in pharmaceutical formulations
and the quantitative analysis of chloropheniramine maleate and
phenylephrine hydrochloride in nasal drops also reported by Nevin
Erk,2000.
Determination of hydrochlorothiazide and amiloride hydrochloride was
reported by Murat Katal and Nevin Erk, 1999. It has also been reported
that the determination of benazepril hydrochloride and
hydrochlorothiazide can be achieved using second derivative ( Alaa El-
Gindy et. Al, 2001). Second derivative spectrophotometry has been

applied for the simultaneous determination of vitamin C and dipyrone (T.
Aburajai et. Al,2000). Derivative spectroscopy has also been employed
for the determination of enoxacin and nalidixic acid in tablets (H.
Hopkala and D. Kowalczuk, 2000). The determination of Amlodipine-
Enalapril Maleate and Amlodipine –Lisinopril in tablets has also been
accomplished by derivative spectroscopy (C. V. N. Prasad, et, al,1999).
The application of derivative spectroscopy as a stability indicating
method for the study of degradation and decomposition of drugs during
stability studies has been reported by D. Castro et, al, 1999, for the
determination of omeprazole tablet in aqueous solution. Analysis of
Acebutolol HCl in presence of it is acid- induced degradation products,
has been reported by Alaa El-Gindy et, al. Similar work has been reported
for the determination of Benoxinate hydrochloride and its degradation
product by first derivative spectrophotometry (Alaa El-Gindy).
Derivative spectroscopy has been applied in the determination of drugs in
pharmaceutical dosage form such as creams and ointments. It has
reported that Micanazole in pharmaceutical creams can determined by
derivative spectroscopy without prior extraction of active ingredient
(Nilgun Gunden Goger and Lerzan Gokcen).
Derivative spectroscopy has reported for the analysis of drugs in
biological fluids (Alaa El-Gindy et. al). The determination of cefuroxime

and cefadroxil in urine is an example of such application of derivative
spectroscopic analysis.
The technique has also been reported (E. M. Hassan et. al) for the
determination of cisapride in pharmaceutical preparations.
Application of second derivative spectrometric has also been employed
in the determination of simvastatin in tablet dosage form ( Lei Wang and
Mandana Asgharnejad). Durisehvar Ozer and Hulya Senel have applied
derivative spectrophotometry for the determination of lisinopril in
pharmaceutical preparations.
In the present work, a derivative spectroscopy method has been
established for analysis of chloroquine phosphate. The method has been
found to be precise accurate and selective. It was employed for analysis
of chloroquine phosphate in different pharmaceutical dosage forms. The
results obtained showed that the developed method is similar in
selectivity to the HPLC method and superior to the zero order and the
official non-aqueous titration methods. While it has, advantage over the
HPLC method being less time consuming and less expensive.
The derivative method found to be suitable for assay of chloroquine
phosphate in plasma. This will likely make further future biological work
with chloroquine phosphate to be meaningful.

5.2. Recommendation:
5.2.1. The present method is suitable as a routine analytical procedure for
analysis of chloroquine phosphate in different pharmaceutical dosage
forms.
5.2.2. The method can further employed for analysis of chloroquine
phosphate in presence of its metabolite i.e. in biological fluids.

5.2.3. Further work recommended for employing this method as a
stability indicating method for the study of chloroquine phosphate in
presence of its decomposition and degradation products.
5.2.4. Work should been extended for studying drug combinations.
5.2.5. The method should been applied in determination of drugs in other
pharmaceutical dosage form such as creams and ointments without prior
extraction of active ingredients.
5.2.6. The derivative spectroscopic method should been applied in the
assay of different pharmaceutical preparations, as it is simple, rapid,
precise and accurate, less coasty and less time consuming than either the
official non-aqueous titremetric method or the HPLC method.

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Appendix
Table 13: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Starch:
Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv.
No HPLC
221nm 225 nm 236 nm 239nm 256nm 260nm 331nm 333nm 343nm 349nm
1 100.9 104.1 99.5 97.1 99.6 97.1 99.7 100 100.8 97.7 100.5
2 104 105.3 104 100 104.2 103 103.7 97.1 103.4 104.9 103.3
3 103.2 103.5 102.5 101.9 103.2 102.3 102.7 101.8 102.2 102.9 101.8
Table14: Statistical analysis of the results of Chloroquine Phosphate
(%w/w) in presence of starch:

t-test F-test
(experimental) (experimental)
Pair 1 Drv.225nm –Abs.221nm 1.994 3.5392
Pair 2 Drv.225nm – HPLC 4.126 17.0256
Pair 3 Drv.239nm–Abs. 236nm 2.376 5.6452
Pair 4 Drv.239nm – HPLC -1.912 3.6574
Pair 5 Drv.260nm–Abs.256nm 3.123 9.7512
Pair 6 Drv.260nm – HPLC 0.897 0.8044
Pair 7 Drv.333nm–Abs.331nm 1.128 1.2715
Pair 8 Drv.333nm – HPLC 1.123 1.2613
Pair 9 Drv.349nm–Abs.343nm 0.211 0.0447
Pair 10 Drv.349nm – HPLC 0.024 0.0006
The theoretical F at the 95% confidence level and degree of freedom Ø1 = 2 Ø2 =2 (19.0)
While the theoretical t at the 95% confidence level and degree of freedom Ø1 = 2 Ø2 =2 (4.303).
Table 15: Results of analysis of Chloroquine Phosphate (%w/w) in presence aerosil
No HPLC
221nm 225nm 236nm 239nm 256nm 260nm 331nm 333nm 343nm 349nm
1 101.3 103.4 99.7 100 99.8 97.1 100.3 100 101.7 98.8 99.9
2 101.3 102.9 101.4 97.1 101.6 100 102.3 100 102 101.7 100.2
3 102.9 101.9 99.7 99.5 100.5 98.5 101 99.5 101.2 100.5 99.5

(%w/w) in presence of Aerosil:
t-test F-test
(experimental) (experimental))
Pair 1 Drv.225nm –Abs.221nm -0.937 0.8773

* *
Pair 2 Drv.225nm – HPLC 8.665 75.088
Pair 3 Drv.239nm–Abs. 236nm 0.961 0.9231
Pair 4 Drv.239nm – HPLC -0.952 0.9069
Pair 5 Drv.260nm–Abs.256nm 6.533 42.677
Pair 6 Drv.260nm – HPLC -1.754 3.0768
Pair 7 Drv.333nm–Abs.331nm 2.352 5.5296
Pair 8 Drv.333nm – HPLC -0.423 0.1791
Pair 9 Drv.349nm–Abs.343nm 1.608 2.5867
Pair 10 Drv.349nm – HPLC -0.937 0.3372
*
significant difference
Table 17: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Avicel
No HPLC
1 101.1 100 101.3 97.1 101.4 100 99.7 100 103 102.5 103.2
2 101.5 101.6 101.6 100 101.9 103 103.5 102.7 102.8 103.7 102.8
3 100.9 101.5 101.9 98.6 101.5 101.5 101.7 101.3 102.5 103.1 103.3

(%w/w) in presence of Avicel:
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.300 0.090
Pair 2 Drv.225nm – HPLC 2.000 4.000
Pair 3 Drv.239nm–Abs. 236nm 3.979 15.834
Pair 4 Drv.239nm – HPLC 4.741 22.474
Pair 5 Drv.260nm–Abs.256nm 0.138 0.019
Pair 6 Drv.260nm – HPLC 1.622 2.630
Pair 7 Drv.333nm–Abs.331nm 0.933 0.871
Pair 8 Drv.333nm – HPLC 1.958 3.832
Pair 9 Drv.349nm–Abs.343nm 0.543 0.295
Pair 10 Drv.349nm – HPLC 0.127 0.016
Table 19: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Mg Sterate
No HPLC
1 104.10 102.20 102.90 101.50 101.5 100.0 101.60 100.90 101.60 101.90 99.90
2 103.30 102.30 102.30 102.90 102.1 99.50 102.40 100.30 102.10 101.20 101.2
3 104.50 103.90 103.50 101.20 100.1 101.5 101.90 101.90 101.90 100.10 100.9

(%w/w) in presence of Mg Sterate:
t-test F-test
(experimental) (experimental
Pair 1 Drv.225nm –Abs.221nm 3.035 9.211
Pair 2 Drv.225nm – HPLC 3.845 14.787
Pair 3 Drv.239nm–Abs. 236nm 1.206 1.454
Pair 4 Drv.239nm – HPLC 2.661 2.082
Pair 5 Drv.260nm–Abs.256nm 0.754 0.569
Pair 6 Drv.260nm – HPLC 0.477 0.228
Pair 7 Drv.333nm–Abs.331nm 1.512 2.286
Pair 8 Drv.333nm – HPLC 0.579 0.335
Pair 9 Drv.349nm–Abs.343nm 1.315 1.730
Pair 10 Drv.349nm – HPLC 0.480 0.231
Table 21: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Lactose
No HPLC
1 101.60 100.00 102.50 100.00 102.30 103.0 102.20 100.90 102.10 100.00 101.2
2 101.20 102.20 99.70 100.50 100.90 100.5 100.80 100.50 101.70 102.90 100.5
3 101.50 101.50 101.50 101.50 101.20 101.9 103.20 99.30 101.50 101.50 101.5

(%w/w) in presence of Lactose:
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.264 0.070
Pair 2 Drv.225nm – HPLC 0.198 0.039
Pair 3 Drv.239nm–Abs. 236nm 0.570 0.325
Pair 4 Drv.239nm – HPLC 1.000 1.000
Pair 5 Drv.260nm–Abs.256nm 0.909 0.826
Pair 6 Drv.260nm – HPLC 1.344 1.806
Pair 7 Drv.333nm–Abs.331nm 1.709 2.920
Pair 8 Drv.333nm – HPLC 1.210 1.464
Pair 9 Drv.349nm–Abs.343nm 0.311 0.097
Pair 10 Drv.349nm – HPLC 0.378 0.143
Table 23: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Talc
No HPLC
1 99.70 102.60 98.90 100.00 99.10 97.90 99.40 100.00 101.40 102.50 102.2
2 103.10 102.70 103.10 97.10 103.30 103.0 104.10 98.50 103.40 97.70 98.80
3 101.40 101.80 101.50 102.90 101.20 101.8 101.70 103.20 102.20 103.20 103.1

(%w/w) in presence of Talc:
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.973 0.946
Pair 2 Drv.225nm – HPLC 0653 0.427
Pair 3 Drv.239nm–Abs. 236nm 0.482 0.233
Pair 4 Drv.239nm – HPLC 2.274 5.172
Pair 5 Drv.260nm–Abs.256nm 0.577 0.333
Pair 6 Drv.260nm – HPLC 0.188 0.035
Pair 7 Drv.333nm–Abs.331nm 0.523 0.273
Pair 8 Drv.333nm – HPLC 1.128 1.272
Pair 9 Drv.349nm–Abs.343nm 0.533 0.284
Pair 10 Drv.349nm – HPLC 0.534 0.285
Table 25: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Acacia
No HPLC
1 99.80 97.80 99.10 97.10 99.30 97.10 100.50 100.50 100.10 100.00 98.90
2 98.60 100.20 100.50 102.90 100.90 100.0 100.30 99.80 101.30 98.80 100.2
3 99.50 99.20 99.80 101.10 100.10 99.80 100.40 101.20 100.70 102.20 100.9
Table 26: Statistical analysis of the results of Chloroquine Phosphate

(% w/w) in presence of Acacia:
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.224 0.050
Pair 2 Drv.225nm – HPLC 1.875 3.516
Pair 3 Drv.239nm–Abs. 236nm 0.429 0.184
Pair 4 Drv.239nm – HPLC 0.282 0.079
Pair 5 Drv.260nm–Abs.256nm 2.021 4.085
Pair 6 Drv.260nm – HPLC 2.231 4.979
Pair 7 Drv.333nm–Abs.331nm 0.264 0.070
Pair 8 Drv.333nm – HPLC 0.853 0.728
Pair 9 Drv.349nm–Abs.343nm 0.315 0.100
Pair 10 Drv.349nm – HPLC 0.384 0.147
Table 27: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Methyl paraben
No HPLC
1 109.90 99.70 122.70 47.10 162.00 124.2 101.70 103.30 102.20 100.00 98.90
2 109.90 102.70 122.40 50.00 164.50 120.9 98.90 102.60 101.10 99.80 99.50
3 108.70 101.50 121.50 48.90 163.90 122.8 102.10 99.10 99.50 99.30 100.3

(%w/w) in presence of Methyl paraben
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 8.200 67.24
Pair 2 Drv.225nm – HPLC 2.335 5.452
Pair 3 Drv.239nm–Abs. 236nm 71.050 5048.2
Pair 4 Drv.239nm – HPLC 71.75 5147.3
Pair 5 Drv.260nm–Abs.256nm 24.311 591.03
Pair 6 Drv.260nm – HPLC 19.869 394.78
Pair 7 Drv.333nm–Abs.331nm 0.387 0.154
Pair 8 Drv.333nm – HPLC 1.241 1.540
Pair 9 Drv.349nm–Abs.343nm 2.133 4.548
Pair 10 Drv.349nm – HPLC 0.218 0.0475
Table 29: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Propyl paraben
No HPLC
1 106.40 102.70 113.70 61.80 143.50 114.3 98.90 97.50 97.50 96.70 97.40
2 107.20 99.10 112.50 60.50 141.20 113.0 99.50 98.20 99.20 98.90 99.20
3 106.10 101.90 112.00 62.20 139.50 114.5 97.90 99.50 98.20 97.50 98.50

(%w/w) in presence of Propyl paraben
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 3.835 14.704
Pair 2 Drv.225nm – HPLC 1.813 3.286
Pair 3 Drv.239nm–Abs. 236nm 71.430 5102.31
Pair 4 Drv.239nm – HPLC 39.28 1542.205
Pair 5 Drv.260nm–Abs.256nm 21.684 470.205
Pair 6 Drv.260nm – HPLC 16.907 285.8
Pair 7 Drv.333nm–Abs.331nm 0.373 0.139
Pair 8 Drv.333nm – HPLC 0.058 0.0033
Pair 9 Drv.349nm–Abs.343nm 3.928 15.427
Pair 10 Drv.349nm – HPLC 3.288 10.811
Table 31:Results of analysis of Chloroquine Phosphate (%w/w) in presence of Dicalcium phosphate
No HPLC
1 101.70 101.40 100.70 97.10 102.80 103.0 99.80 100.20 100.60 102.50 101.5
2 102.90 103.90 102.10 97.80 102.00 100.0 101.90 97.40 100.40 97.70 98.50
3 99.10 98.60 98.50 101.90 99.20 98.60 98.90 101.20 99.80 100.90 100.8

(%w/w) in presence of Dicalcium phosphate
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.142 0.020
Pair 2 Drv.225nm – HPLC 0.456 0.208
Pair 3 Drv.239nm–Abs. 236nm 0.610 0.372
Pair 4 Drv.239nm – HPLC 0.824 0.678
Pair 5 Drv.260nm–Abs.256nm 1.244 1.548
Pair 6 Drv.260nm – HPLC 0.216 0.047
Pair 7 Drv.333nm–Abs.331nm 0.296 0.088
Pair 8 Drv.333nm – HPLC 1.243 1.544
Pair 9 Drv.349nm–Abs.343nm 0.070 0.005
Pair 10 Drv.349nm – HPLC 0.192 0.037
Table 33: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Titanium Dioxide
No HPLC
1 102.70 102.80 103.00 100.00 106.87 106.3 106.70 101.50 106.90 105.80 101.3
2 104.60 103.80 105.80 102.50 106.70 103.0 105.60 104.90 105.00 104.90 104.1
3 101.90 102.20 102.50 106.10 103.10 105.9 102.90 105.50 103.00 102.90 103.3
(%w/w) in presence of Titanium Dioxide
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.394 0.155
Pair 2 Drv.225nm – HPLC 0.043 0.002
Pair 3 Drv.239nm–Abs. 236nm 0.400 0.160
Pair 4 Drv.239nm – HPLC 0.023 0.001
Pair 5 Drv.260nm–Abs.256nm 0.261 0.068
Pair 6 Drv.260nm – HPLC 1.221 1.491
Pair 7 Drv.333nm–Abs.331nm 0.487 0.237
Pair 8 Drv.333nm – HPLC 1.800 3.241
Pair 9 Drv.349nm–Abs.343nm 1.300 1.690
Pair 10 Drv.349nm – HPLC 1.108 1.227
Table 35: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Opadry
No HPLC
1 99.50 98.60 102.50 102.90 103.10 100.00 100.10 103.70 104.90 100.00 101.7
2 98.50 101.50 100.50 99.50 101.90 102.80 104.90 98.90 100.60 102.90 101.2
3 101.20 99.30 99.20 100.20 98.70 97.80 98.50 101.20 99.50 99.80 99.90

(%w/w) in presence of Opadry
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.045 0.002
Pair 2 Drv.225nm – HPLC 1.114 1.242
Pair 3 Drv.239nm–Abs. 236nm 0.225 0.051
Pair 4 Drv.239nm – HPLC 0.078 0.006
Pair 5 Drv.260nm–Abs.256nm 0.893 0.798
Pair 6 Drv.260nm – HPLC 0.626 0.391
Pair 7 Drv.333nm–Abs.331nm 0.033 0.001
Pair 8 Drv.333nm – HPLC 0.250 0.063
Pair 9 Drv.349nm–Abs.343nm 0.357 0.128
Pair 10 Drv.349nm – HPLC 0.034 0.001
Table 37: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Propylene Glycol
No HPLC
1 100.50 100.90 99.70 100.00 100.10 97.10 99.70 98.50 100.70 97.70 101.1
2 101.20 102.80 101.50 101.20 101.30 101.5 101.90 101.20 99.50 100.90 99.50
3 99.50 99.10 100.90 98.90 99.30 100.2 100.80 97.90 101.50 101.50 100.3
Table38: Statistical analysis of the Percentage results of Chloroquine Phosphate

(%w/w) in presence of Propylene Glycol
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.918 0.842
Pair 2 Drv.225nm – HPLC 0.464 0.216
Pair 3 Drv.239nm–Abs. 236nm 0.968 0.937
Pair 4 Drv.239nm – HPLC 0.270 0.073
Pair 5 Drv.260nm–Abs.256nm 0.528 0.278
Pair 6 Drv.260nm – HPLC 0.398 0.159
Pair 7 Drv.333nm–Abs.331nm 2.403 5.774
Pair 8 Drv.333nm – HPLC 0.785 0.616
Pair 9 Drv.349nm–Abs.343nm 0.411 0.169
Pair 10 Drv.349nm – HPLC 0.170 0.029
Table 39: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Glycerin
No HPLC
1 99.20 100.00 100.40 97.10 101.70 97.10 104.60 103.40 101.70 98.80 99.20
2 101.60 102.60 99.00 100.00 100.90 103.0 102.50 97.80 101.90 101.20 101.6
3 100.20 99.30 101.40 101.00 99.50 100.5 99.11 101.20 99.20 100.90 100.2

(%w/w) in presence of Glycerin
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.498 0.248
Pair 2 Drv.225nm – HPLC 0.684 0.467
Pair 3 Drv.239nm–Abs. 236nm 0.711 0.505
Pair 4 Drv.239nm – HPLC 0.608 0.37
Pair 5 Drv.260nm–Abs.256nm 0.241 0.058
Pair 6 Drv.260nm – HPLC 0.041 0.002
Pair 7 Drv.333nm–Abs.331nm 0.648 0.42
Pair 8 Drv.333nm – HPLC 0.268 0.072
Pair 9 Drv.349nm–Abs.343nm 0.477 0.227
Pair 10 Drv.349nm – HPLC 0.035 0.001
Table 41: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Sucrose
No HPLC
1 99.10 97.50 101.70 100.00 99.70 97.10 102.20 103.20 101.00 102.50 99.10
2 101.20 102.60 99.10 100.00 102.10 103.0 99.20 101.50 101.90 97.70 101.2
3 100.50 100.20 100.20 101.20 100.10 100.2 100.50 97.10 99.50 100.20 100.5

(%w/w) in presence of Sucrose
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.192 0.037
Pair 2 Drv.225nm – HPLC 0.104 0.011
Pair 3 Drv.239nm–Abs. 236nm 0.075 0.006
Pair 4 Drv.239nm – HPLC 0.172 0.030
Pair 5 Drv.260nm–Abs.256nm 0.504 0.254
Pair 6 Drv.260nm – HPLC 0.089 0.008
Pair 7 Drv.333nm–Abs.331nm 0.019 0.0004
Pair 8 Drv.333nm – HPLC 0.249 0.062
Pair 9 Drv.349nm–Abs.343nm 0.374 0.14
Pair 10 Drv.349nm – HPLC 0.056 0.003
Table 43: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Saccharin Na
No HPLC
1 161.80 159.50 205.10 147.50 106.30 97.80 102.80 101.50 103.60 102.50 103.2
2 158.50 160.10 207.00 150.00 104.60 99.80 103.50 103.90 102.70 103.10 101.5
3 160.20 161.20 205.60 148.90 105.20 101.2 102.50 102.40 101.50 101.90 102.4

(%w/w) in presence of Saccharin Na
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.082 0.007
Pair 2 Drv.225nm – HPLC 72.187 5211
Pair 3 Drv.239nm–Abs. 236nm 215.818 46577
Pair 4 Drv.239nm – HPLC 38.283 1465
Pair 5 Drv.260nm–Abs.256nm 4.161 17.13
Pair 6 Drv.260nm – HPLC 2.089 4.363
Pair 7 Drv.333nm–Abs.331nm 0.661 0.437
Pair 8 Drv.333nm – HPLC 0.196 0.039
Pair 9 Drv.349nm–Abs.343nm 0.200 0.040
Pair 10 Drv.349nm – HPLC 0.181 0.033
Table 45: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Sodium Citrate
No HPLC
1 102.60 103.90 102.60 100.00 102.30 103.0 102.10 104.50 101.10 101.50 101.9
2 103.30 99.20 102.20 98.50 102.60 100.2 104.00 101.50 102.50 98.80 99.90
3 99.80 104.20 99.80 101.90 100.20 99.50 98.50 99.60 99.50 102.52 102.4

(%w/w) in presence of Sodium Citrate
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.215 0.046
Pair 2 Drv.225nm – HPLC 1.190 1.415
Pair 3 Drv.239nm–Abs. 236nm 0.787 0.620
Pair 4 Drv.239nm – HPLC 3.092 9.563
Pair 5 Drv.260nm–Abs.256nm 0.893 0.797
Pair 6 Drv.260nm – HPLC 0.409 0.167
Pair 7 Drv.333nm–Abs.331nm 0.227 0.052
Pair 8 Drv.333nm – HPLC 0.281 0.079
Pair 9 Drv.349nm–Abs.343nm 0.048 0.002
Pair 10 Drv.349nm – HPLC 1.301 1.694
Table 47: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Citric Acid
No HPLC
1 99.90 97.40 102.20 100.00 102.50 103.0 103.60 101.90 103.60 104.20 100.6
2 100.20 101.20 100.50 101.20 101.30 101.4 101.30 100.50 99.20 99.50 101.2
3 98.90 100.30 99.50 99.80 98.90 98.50 99.20 99.50 100.50 100.10 99.90

(%w/w) in presence of Citric Acid
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.027 0.007
Pair 2 Drv.225nm – HPLC 0.819 0.671
Pair 3 Drv.239nm–Abs. 236nm 0.441 0.194
Pair 4 Drv.239nm – HPLC 1.257 1.581
Pair 5 Drv.260nm–Abs.256nm 0.256 0.066
Pair 6 Drv.260nm – HPLC 0.363 0.132
Pair 7 Drv.333nm–Abs.331nm 1.268 1.608
Pair 8 Drv.333nm – HPLC 0.107 0.012
Pair 9 Drv.349nm–Abs.343nm 0.563 0.317
Pair 10 Drv.349nm – HPLC 0.452 0.204
Table 49: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Sorbitol
No HPLC
1 102.30 100.00 103.50 102.90 99.50 103.0 99.90 102.70 101.60 103.70 101.9
2 98.90 101.90 99.30 99.50 103.80 97.10 105.30 100.00 102.60 100.00 99.50
3 100.20 100.00 100.50 100.00 100.90 100.2 100.10 99.50 100.50 99.80 100.9

(%w/w) in presence of Sorbitol
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.108 0.012
Pair 2 Drv.225nm – HPLC 0.103 0.011
Pair 3 Drv.239nm–Abs. 236nm 1.192 1.421
Pair 4 Drv.239nm – HPLC 0.061 0.004
Pair 5 Drv.260nm–Abs.256nm 0.439 0.193
Pair 6 Drv.260nm – HPLC 0.660 0.435
Pair 7 Drv.333nm–Abs.331nm 0.440 0.194
Pair 8 Drv.333nm – HPLC 0.048 0.002
Pair 9 Drv.349nm–Abs.343nm 0.293 0.086
Pair 10 Drv.349nm – HPLC 0.477 0.228
Table 51: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Xanthan Gum
No HPLC
1 97.10 97.10 98.70 100.00 102.20 97.10 99.40 104.10 100.60 102.50 102.6
2 99.80 101.40 102.10 97.10 99.10 100.0 99.00 100.00 104.40 99.90 101.6
3 100.50 100.44 100.20 101.10 100.40 101.5 104.10 99.50 98.50 101.40 99.80

(%w/w) in presence of Xanthan Gum
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.944 0.892
Pair 2 Drv.225nm – HPLC 0.878 0.770
Pair 3 Drv.239nm–Abs. 236nm 0.458 0.210
Pair 4 Drv.239nm – HPLC 1.132 1.283
Pair 5 Drv.260nm–Abs.256nm 0.508 0.258
Pair 6 Drv.260nm – HPLC 0.865 0.748
Pair 7 Drv.333nm–Abs.331nm 0.136 0.018
Pair 8 Drv.333nm – HPLC 0.148 0.022
Pair 9 Drv.349nm–Abs.343nm 0.043 0.002
Pair 10 Drv.349nm – HPLC 0.070 0.005
Table 53: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Tween 80
No HPLC
1 100.10 101.80 102.80 102.50 103.50 100.0 103.20 100.10 104.40 101.80 103.2
2 100.10 103.20 100.40 102.10 100.70 104.1 101.90 104.10 101.90 100.00 101.2
3 102.50 100.00 101.20 99.50 99.50 98.60 99.50 98.50 98.80 104.90 98.50
(%w/w) in presence of Tween 80

t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.456 0.208
Pair 2 Drv.225nm – HPLC 0.660 0.436
Pair 3 Drv.239nm–Abs. 236nm 0.101 0.010
Pair 4 Drv.239nm – HPLC 0.726 0.528
Pair 5 Drv.260nm–Abs.256nm 0.166 0.027
Pair 6 Drv.260nm – HPLC 0.038 0.001
Pair 7 Drv.333nm–Abs.331nm 0.411 0.169
Pair 8 Drv.333nm – HPLC 0.038 0.002
Pair 9 Drv.349nm–Abs.343nm 0.191 0.037
Pair 10 Drv.349nm – HPLC 0.456 0.243
Table 55: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Gelatin
No HPLC
1 101.30 100.00 102.80 102.00 103.50 103.1 103.90 100.00 101.40 97.70 99.30
2 100.10 103.90 100.40 102.90 100.60 100.3 100.20 101.50 102.50 104.90 102.6
3 102.20 99.80 99.50 98.90 99.30 100.0 99.10 102.50 98.90 100.50 101.9

(%w/w) in presence of Gelatin
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.017 0.0003
Pair 2 Drv.225nm – HPLC 0.032 0.001
Pair 3 Drv.239nm–Abs. 236nm 0.343 0.118
Pair 4 Drv.239nm – HPLC 0.000 0.000
Pair 5 Drv.260nm–Abs.256nm 0.000 0.000
Pair 6 Drv.260nm – HPLC 0.068 0.005
Pair 7 Drv.333nm–Abs.331nm 0.123 0.015
Pair 8 Drv.333nm – HPLC 0.114 0.013
Pair 9 Drv.349nm–Abs.343nm 0.052 0.003
Pair 10 Drv.349nm – HPLC 0.184 0.034
Table 57: Results of analysis of Chloroquine Phosphate (%w/w) in presence of Phosphoric Acid
No HPLC
1 101.50 101.30 99.90 102.90 102.40 103.0 103.70 100.00 103.40 103.10 100.1
2 98.50 100.00 101.90 100.00 100.30 97.10 101.10 103.20 102.50 100.00 101.9
3 100.20 99.20 100.20 98.50 99.50 101.2 98.90 99.20 98.50 99.50 100.5
(%w/w) in presence of Phosphoric Acid
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.136 0.018
Pair 2 Drv.225nm – HPLC 0.702 0.493
Pair 3 Drv.239nm–Abs. 236nm 0.125 0.016
Pair 4 Drv.239nm – HPLC 0.232 0.054
Pair 5 Drv.260nm–Abs.256nm 0.202 0.041
Pair 6 Drv.260nm – HPLC 0.175 0.031
Pair 7 Drv.333nm–Abs.331nm 0.253 0.064
Pair 8 Drv.333nm – HPLC 0.044 0.002
Pair 9 Drv.349nm–Abs.343nm 0.587 0.345
Pair 10 Drv.349nm – HPLC 0.022 0.001
Table 59: Results of analysis of Chloroquine Phosphate (%w/w) in presence of
disintegrants used in the formulations
Method Absorbance Derivative HPLC
Wavelength 221nm 236nm 256nm 331nm 343nm 225nm 239nm 260nm 333nm 349nm 349nm
Starch 102.7 102 102.19 102.03 102.1 104.3 99.7 100.8 99.6 101.8 101.9
Aerosil 101.8 100.3 100.6 101.2 101.6 102.7 98.9 98.5 99.8 100.3 99.9
Avicel 101.67 101.6 101.6 101.63 102.77 101.37 98.57 101.5 101.33 103.03 103.1

Lubricant used in the formulations

Mg
103.97 102.9 101.23 101.97 101.87 102.8 101.77 100.33 101.03 101.07 100.67
Sterate
Talc 101.4 101.2 101.2 101.7 102.3 102.4 100 100.9 100.6 101.1 101.4
Diluents used in the formulations

Lactose 101.43 101.23 101.47 102.07 101.77 101.23 100.67 101.8 100.2 101.47 101.07
Dical. Phos. 101.2 100.4 101.3 100.2 100.3 101.3 98.9 100.5 99.6 100.4 100.77
Sucrose 100.3 100.3 100.6 100.6 100.8 100.1 100.4 100.1 100.6 100.1 100.61

Coloring agent used in the Formulations

Wave length 221nm 236nm 256nm 331nm 343nm 225nm 29nm 260nm 333nm 349nm 349nm
Titanium dioxide 103.1 103.8 105.6 105.1 105 102.9 102.9 105.1 104 104.5 102.9
Opadry 99.7 100.7 101.2 101.2 101.7 99.8 100.9 100.2 101.3 100.9 101.8
pH adjustment excipent used in the Formulations

Sodium Citrate 101.9 101.5 101.7 100.5 101 102.4 100.1 100.9 101.9 100.9 101.75
Citric Acid 99.7 100.7 100.9 101.4 101.1 99.6 100.3 101 100.6 101.3 100.6
Phosphoric Acid 100.1 100.7 100.7 101.2 101.5 100.2 100.5 100.4 100.8 100.9 101.4

Anti-microbial agents used in the Formulations

Methyl paraben 109.5 122.2 163.5 100.9 100.9 109.5 122.2 163.5 100.9 100.9 99.6
Propyl paraben 106.6 112.7 141.4 98.8 98.3 100.9 100 99.6 99.2 100 98.4
Propylene glycol 100.4 100.7 100.2 100.8 100.6 100.9 100 99.6 99.2 100 100.3
Glycerin 100.3 100.3 100.7 102.1 100.9 100.6 99.4 100.2 100.8 100.32 100.3
Saccharin sodium 160.2 205.9 105.4 102 102.6 160.3 148.8 99.6 102.6 102.5 102.4
Emulsifying agent used in the Formulations

Acacia 99.3 99.8 100.1 100.4 100.7 99.1 100.4 99 100.5 100.3 100
Gelatin 101.2 100.9 101.3 101.1 100.9 101.2 101.3 101.1 101.3 101 101.3
Sorbitol 100.5 101.1 101.4 101.8 101.6 100.6 100.8 100.1 100.7 101.2 100.8
Xanthan gum 99.1 100.3 100.6 100.8 101.2 99.6 99.4 99.5 101.2 101.3 101.3
Tween 80 100.9 101.5 101.2 101.5 101.7 101.7 101.4 100.9 100.9 102.2 101
Table 66: Results of analysis of Chloroquine Phosphate injection (%w/w)
Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Non Aqueous
No. HPLC
221nm 225nm 236nm 239nm 256nm 260nm 331nm 333nm 343nm 349nm Titration
1 101.6 101.1 101.9 102.60 102.40 102.80 103.70 101.9 102.3 101.7 101.6 102.2
2 101.5 101.8 101.5 102.10 101.90 100.90 102.90 102.5 101.9 102.2 100.9 101.6
3 101.2 101.5 101.7 101.50 101.60 101.90 103.20 101.7 102.1 101.5 101.8 102.6
Table 67: Statistical analysis of the results of Chloroquine Phosphate injection (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.125 0.016
Pair 2 Drv.225nm – HPLC 0.076 0.006
Pair 3 Drv.225 nm- Non-Aqueous Titration 1.538 2.367
Pair 4 Drv.239 nm –Abs236 nm 1.287 1.658
Pair 5 Drv.239 nm – HPLC 1.347 1.814
Pair 6 Drv.239 nm – Non-Aqueous Titration 0.129 0.017
Pair 7 Drv.260 nm–Abs256 nm 0.222 0.049
Pair 8 Drv.260 nm – HPLC 1.127 1.271
Pair 9 Drv.260 nm – Non -Aqueous Titration 0.615 0.379
Pair 10 Drv.333 nm–Abs331 nm 2.898 8.399
Pair 11 Drv.333 nm – HPLC 1.169 1.367
Pair 13 Drv.343 nm–Abs349 nm 1.000 1.000
Pair 14 Drv.349 nm – HPLC 0.763 0.582
Table 68: Results of analysis of Balsaquine injection (%w/w)
Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Non Aqueous
No. 260n HPLC
221nm 225nm 236nm 239nm 256nm 331nm 333nm 343nm 349nm Titration
m
1 101.7 101.5 100.9 101.90 101.5 99.70 101.50 102.10 101.80 102.50 102.2 100.6
2 99.80 100.9 99.50 100.20 100.3 101.90 99.90 100.50 102.10 100.90 101.9 101.9
3 100.3 101.8 100.5 100.90 100.6 100.50 100.80 99.50 101.50 102.30 101.4 101.5
Table 69: Statistical analysis of the results of Balsaquine injection (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 1.559 2.430
Pair 2 Drv.225nm – HPLC 1.018 1.037
Pair 3 Drv.225 nm & Non-Aqueous Titration 0.189 0.036
Pair 4 Drv.239 nm –Abs236 nm 4.041 16.333
Pair 5 Drv.239 nm – HPLC 1.906 3.634
Pair 7 Drv.260 nm–Abs256 nm 0.102 .010
Pair 8 Drv.260 nm – HPLC 1.550 2.403
Pair 10 Drv.333 nm–Abs331 nm 0.053 0.003
Pair 11 Drv.333 nm – HPLC 2.113 4.463
Pair 13 Drv.343 nm–Abs349 nm 0.154 0.024
Pair 14 Drv.349 nm – HPLC 0.119 0.014
Table 70: Results of analysis of Efroquine tablet (%w/w)
Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Non-Aqueous
No. 236n 239n 331n HPLC
221nm 225nm 256nm 260nm 333nm 343nm 349nm Titration
m m m
1 98.80 97.20 98.80 97.10 97.80 97.20 98.2 96.80 98.00 97.50 97.2 99.0
2 98.20 98.80 98.30 98.80 98.50 97.80 97.9 98.10 98.50 98.30 98.5 98.7
3 98.50 98.70 98.10 98.50 98.00 98.20 98.6 97.50 98.70 98.50 98.8 99.1
Table 71: Statistical analysis of the results of Efroquine tablet analyzed (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.394 0.155
Pair 2 Drv.225nm – HPLC 0.555 0.308
Pair 4 Drv.239 nm –Abs236 nm 0.372 0.138
Pair 5 Drv.239 nm – HPLC 0.189 0.036
Pair 7 Drv.260 nm–Abs256 nm 1.287 1.658
Pair 8 Drv.260 nm – HPLC 1.982 3.930
Pair 10 Drv.333 nm–Abs331 nm 1.561 2.438
Pair 11 Drv.333 nm – HPLC 2.333 5.444
Pair 13 Drv.343 nm–Abs349 nm 3.000 9.000
Pair 14 Drv.349 nm – HPLC 0.359 0.129
Table 72: Results of analysis of Chloroquine Phosphate tablet (%w/w)
No. HPLC
1 106.40 103.90 104.10 105.90 104.20 103.90 106.40 103.90 104.10 105.90 104.20 103.90
2 105.80 104.50 105.90 104.20 106.20 105.90 105.80 104.50 105.90 104.20 106.20 105.90
3 104.90 104.10 106.50 105.80 105.80 105.70 104.90 104.10 106.50 105.80 105.80 105.70
Table 73: Statistical analysis of the results of Chloroquine Phosphate table analyzed (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 3.040 9.240
Pair 2 Drv.225nm – HPLC 0.693 0.481
Pair 4 Drv.239 nm –Abs236 nm 0.192 0.037
Pair 5 Drv.239 nm – HPLC 3.179 10.105
Pair 7 Drv.260 nm–Abs256 nm 3.500 12.250
Pair 8 Drv.260 nm – HPLC 0.756 0.571
Pair 10 Drv.333 nm–Abs331 nm 0.854 0.730
Pair 11 Drv.333 nm – HPLC 3.478 12.094
Pair 13 Drv.343 nm–Abs349 nm 2.897 8.395
Pair 14 Drv.349 nm – HPLC 1.000 1.000
Table 74: Results of analysis of Amiquine tablet (%w/w)
No. HPLC
1 98.70 97.30 98.50 100.00 101.60 100.00 104.10 104.30 101.90 102.30 100.10 100.20
2 102.30 101.90 104.00 102.90 104.70 103.00 104.00 104.20 103.80 101.90 103.10 102.50
3 100.50 100.90 99.50 101.90 98.20 99.80 98.00 97.80 98.20 99.50 99.40 100.50
Table 75: Statistical analysis of the results of Amiquine tablets (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 0.896 0.803

Pair 2 Drv.225nm – HPLC 0.664 0.441
Pair 4 Drv.239 nm –Abs236 nm 0.889 0.791
Pair 5 Drv.239 nm – HPLC 0.830 0.689
Pair 7 Drv.260 nm–Abs256 nm 0.523 0.273
Pair 8 Drv.260 nm – HPLC 0.400 0.160
Pair 10 Drv.333 nm–Abs331 nm 0.500 0.25
Pair 11 Drv.333 nm – HPLC 0.736 0.542
Pair 13 Drv.343 nm–Abs349 nm 0.070 0.005
Pair 14 Drv.349 nm – HPLC 0.370 0.137
Table 76: Results of analysis of Lariago tablet (%w/w)
Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Abs. Drv. Non-
No. 221n 239n 256n HPLC Aqueous
225nm 236nm 260nm 331nm 333nm 343nm 349nm
1 105.6 104.9 106.2 105.9 106.9 106 105.1 103.8 105 104.9 102.9 99.9
2 103.4 103.5 104.5 102.9 104.9 102.9 103.3 105.4 104.2 103.8 102.5 99.6
3 104.7 104.5 105.2 106.5 105.3 105.2 104.8 102.5 103.9 105.1 101.5 99.7
Table 77: Statistical analysis of the results of Lariago tablet (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm 1.143 1.306
Pair 2 Drv.225nm – HPLC 3.464 12.000
Pair 3 Drv.225 nm - Non-Aqueous Titration 0.854 0.73
Pair 4 Drv.239 nm –Abs236 nm 0.238 0.057
Pair 5 Drv.239 nm – HPLC 2.103 4.421
Pair 7 Drv.260 nm–Abs256 nm 1.816 3.297
Pair 8 Drv.260 nm – HPLC 2.365 5.592
Pair 10 Drv.333 nm–Abs331 nm 0.375 0.141
Pair 11 Drv.333 nm – HPLC 2.459 6.047
Pair 13 Drv.343 nm–Abs349 nm 0.475 0.226
Pair 14 Drv.349 nm – HPLC 3.379 11.417
Table 78: Results of analysis of Chloroquine syrup (JPM) (%w/w)
No. HPLC
1 200.90 202.50 110.70 102.20 116.3 99.90 105.80 99.90 103.40 100.50 97.90 99.50
2 198.50 199.50 109.50 101.90 115.40 99.20 105.50 100.50 104.20 99.90 100.20 98.20
3 210.10 208.00 111.40 99.20 113.50 100.50 104.20 101.90 102.90 98.50 99.10 98.9
Table 79: Statistical analysis of the results of Chloroquine syrup (JPM) analyzed (%w/w)
t-test F-test
Pair 1 Drv.225nm –Abs.221nm -0.145 0.021

Pair 2 Drv.225nm – HPLC 37.556* 1410.5*
Pair 3 Drv.225 nm & Non-Aqueous Titration 44.115* 1946.1*
Pair 4 Drv.239 nm –Abs236 nm 6.702* 44.92*
Pair 5 Drv.239 nm – HPLC 1.661 2.760
Pair 7 Drv.260 nm–Abs256 nm 13.799* 190.418*
Pair 8 Drv.260 nm – HPLC 0.873 0.762
Pair 10 Drv.333 nm–Abs331 nm 4.068 16.547
Pair 11 Drv.333 nm – HPLC 2.306 5.319
Pair 13 Drv.343 nm–Abs349 nm 7.986* 63.773*
Pair 14 Drv.349 nm – HPLC 0.555 0.308
* Significant difference
Table 80: Results of analysis of Lariago syrup (%w/w)
No. HPLC
1 142.10 34.30 . 136.90 156.50 106.20 104.40 104.20 105.40 104.70 105.6 98.50
2 136.50 42.10 . 126.50 160.10 104.20 104.80 103.90 102.90 103.90 102.9 97.90
3 139.20 39.50 . 130.50 158.50 102.90 102.20 101.90 103.50 102.50 103.2 100.20
Table 81: Statistical analysis of the results of Lariago syrup analyzed (%w/w)
t-test F-test
* *
Pair 1 Drv.225nm –Abs.221nm 25.828 667.08
* *
Pair 2 Drv.225nm – HPLC 20.847 434.618
* *
Pair 4 Drv.239 nm –Abs236 nm - -
* *
Pair 5 Drv.239 nm – HPLC 12.324 151.887
* *
* *
Pair 7 Drv.260 nm–Abs256 nm 29.654 879.383
Pair 8 Drv.260 nm – HPLC 1.152 1.326
Pair 10 Drv.333 nm–Abs331 nm 2.135 4.558
Pair 11 Drv.333 nm – HPLC -0.723 0.523
Pair 13 Drv.343 nm–Abs349 nm 0.375 0.140
Pair 14 Drv.349 nm – HPLC -0.332 0.110
• Significant difference
Table 82: Results of analysis of Chloroquine Phosphate standard (%w/w) in plasma
by the first derivative and zero absorption spectroscopy
No. Abs. Drv. Abs. Drv.

331nm 333nm 343nm 349nm
1 147.5 99.2 127.9 98.9
2 148.9 100.9 130.2 99.5
3 152.2 99.5 129.5 98.5

Analysis 0f Chloroquine

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‫ﺑﺴﻢ اﻟﻠﺔ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ‬

Analysis 0f Chloroquine Phosphate by

Eltayeb, head department of pharmaceutical chemistry, Faculty of

for his careful supervision.

Also my thanks to professor Mohammed Alzein Haga, Faculty of

his helpful advice and guidance .

My great thanks extended to Balsam Pharmaceutical Company and to

Marrwa Pharmaceutical factory for the allowance to use their quality

control laboratories to do this work and to all the staff of these

Quality Control and Research Directorate and to Ministry of Health for

the financial support of this work.

friends for their assistance and support.

First and before all I thank God.

Table Results of analysis of chloroquine phosphate in presence of xxvii

Table Statistical analysis of the results of chloroquine phosphate xxix

Malaria is one of the most serious protozoal infectious diseases in

man. It is caused infection by the four species of Plasmodium. P.

falciprum causes falciprum (malignant tertian or sub tertian) malaria

individuals if not treated promptly. P. vivax causes vivax (benign

tertian) malaria which is widespread but rarely fatal, although

symptoms during the primary attack can be severe. P. malaria

causes quartan malaria which is generally mild but causes fatal

nephrosis. P. ovale causes ovale (ovale tertian ) malaria which is the

least common type, and produces clinical features similar to P. vivax

(James et al, 1989).

Malaria is a major threat to health and blocks the path to economic

development for individuals, communities and nations.

in Africa, malaria is a problem in every region of the world affecting

both young and old.

killers in tropical Africa. It takes the lives of 1 out of 20 children

pregnant women. It affects the poor and under privileged most

Social, political and economical changes contribute much to the

worsening malaria problem, particularly through large scale

uncontrolled population movements and ecological disturbances

(James et al, 1989).

Construction and environmental changes brought about by

development are creating environment favorable for malaria

transmission. This greatly enhance exacerbating existing problems

opening the way for devastating epidemics in areas which are

previously malaria-free, leading to many deaths and impoverishment

of communities (Bruce-Chwatts et al, 1989).

degrees from holodemisty in the South to hypodemisty in the North. It

mortality which adversely affects the socio-economic development. The

1.1.1. Chemotherapy of malaria:

Antimalarial drugs are classified according to the stage of parasitic

life cycle, which they affect.

Blood schizotocides act on the erythrocytic stages of the parasite,

which are directly responsible for the clinical symptoms of the

disease. They produce clinical cure or suppression of infection by

susceptible strains of all the four species of malaria parasite.

However, since they have no effect on exoerythrocytic forms, they

do not produce a radical cure of relapsing forms of ovale or vivax

Tissue schizotocides act on exoerythrocytic stages of the parasite

cells, or as anti relapse drugs to produce radical cure of vivax and

Gametocyticides destroy the sexual forms of the parasite to interrupt

transmission of the infection to the mosquito vector.

Sporonticides have no direct effect on the gametocytes in the human

host but prevent sporogony in the mosquito.

structure (Susan Budavari et al, 1996) into:

1.1.1.1. quinine salts, which are rapidly acting, blood schizontocides

with some gametocidal activity. They have the chemical structure:

Qinine is available as the following salt:

1.Quinine bisulfate heptahydrate (C20H24N2O2 .H2SO4.7 H2O).