analysis of antimalarials drugs

1. Seminar on methods of analysis of
antimalarials

2. INTRODUCTION
 Malaria is a life-threatening blood disease caused by
parasitic protozoans belonging to
the Plasmodium type transmitted to humans through
the bite of the female Anopheles mosquito.
 Symptoms of malaria are Fever, Chills, Rise in Body
Temperature, Nausea and Vomitting.
 P. falciparum causes the most severe symptoms including
severe anemia, cerebral symptoms, renal failure,
pulmonary edema, and even death.
 Malaria affects approximately 300 million people every
year and results in 1 million deaths wordwide (WHO,
2003).

3. CLASSIFICATION
CLASSIFICATION EXAMPLES
4-aminoquinolines Chloroquine, amodiaquine
Quinoline Mefloquine
Acridine Mepacrine
Cinchona Alkaloid Quinine
Biguanides Proguanil
Diaminopyrimidines Pyrimethamine
8-aminoquinoline Primaquine, bulaquine
Sulfonamides & Sulphones Sulfadiazine, sulfamethopyrazine
Tetracycline Tetracycline, doxicycline
Sesquiterpine lactones Artesunate, artermether

4. 1.CHLOROQUINE PHOSPHATE
SOLUBILITY : Soluble in WATER, Insoluble in
ALCOHOL and CHLOROFORM
MOL. WT : 515.87
PROPERTIES : White, crystalline powder having
bitter test

5. METHOD OF ANALYSIS
A. Thin layer chromatography
B. Non – aqueous titration method
C. Spectrophotometric method
D. Gas – liquid chromatography analysis
E. Identification by spot method

6. (A)THIN LAYER CHROMATOGRAPHY :
 Thin-layer chromatography is performed on a sheet of
glass, plastic, or aluminium foil, which is coated with a
thin layer of adsorbent material, usually silica
gel, aluminium oxide (alumina), or cellulose.
 Genrally percolated silica gel containing a
FLUORESCENCE indicator used.
 The following TLC system are useful for the
identificationas well as the evaluation of the purity of
the drug substances.

7. SYSTEM SPOTTING
SOLUTION
DETECTION Rf VALUE
Methanol
:Water :
Con.
Ammonia
(72:25:3)
Chloroform U.V.-254
Iodine vapour
.28
Chloroform :
Cyclo
Hexane :
Diethylamin
e (5:4:1)
Water U.V.-254
Dragnadroff
reagent
.40
N- Butanol :
Con.
Ammonia :
Alchol
(5:3:2)
Water U.V.-254
Dragnadroff
reagent
.60

8. (B) NON – AQUEOUS TITRATION METHOD :
 Chloroquione Phosphate can also be titrated with acetous 0.1 N
Perchloric acid. The titration may be carried out manually with
CRYSTAL VIOLET as indicator or determine by
POTENTIOMETRICALLY.
 The blank is performed and determine the end point.
 In case of tablet, injection and syrup fist extraction should be carried
out then followed by non – aqueous titration method.
(C) SPECTROPHOTOMETRIC ASSAY :
 The Chloroquione base is obtained by ETHER Or CHLOROFORM
extraction of an alkaline homogenate of the biological sample. After the
separation of interfering materials the base is in turns extracted into a
solution of 0.1N or 0.01N HCl and quantitatively determined by
measuring its U.V. absobance

9.  Absorption maxima are measured at 343, 238, 256 and 222 nm. Measurement
are most favorably made at 343 nm. Where absorption is most intense and least
affected by interfering substance in the biological samples.
.
(D) GAS – LIQUID CHROMATROGRAPHY ANALYSIS :
 When drug in microgram amount from the biological sample after the
solvent extraction, the GLC is used for its determination.
 If the sample on nanogram quantity of Chloroquione phosphate or
base the ECD detector is used.
 The Instrumental condition for GLC are as follows.

10. COLUMN : 3.8 % silicon gum SE 30, 4 ft, glass
DETECTOR : FID
TEMPERATURE: Injection Port : 275 0 C
Column : 240 0 C
Detector : 250 0 C
FLOW RATE : 30 ml/ min Gas --- HELIUM
RETATION TIME : 7.0 MIN

11. (E) IDENTIFICATION BY SPOT METHOD:
TEST COLOUR
Complex with
copper
Pale green
Complex with cobalt Violet
Dimethyl
aminpbenzaldehyde
Yellow
Methyl orange Yellow
H2SO4/ KClO4 Red Violet

12. 2.MEFLOQUINE HYDROCHLORIDE
SOLUBILITY: slightly soluble in WATER. Soluble in ETHANOL &
ETHYL ACETATE
PROPERTIES: odorless and white crystalline powder.
FORMULA: C17H16F6N2O.HCl
MOLACULAR WT: 414.8

13. Method of analysis
 Thin layer chromatography
 High performance chromatography
 U.V. spectroscopy

14. (1)THIN LAYER CHROMATGROPHY:
TLC profiles of bulk drug mefloquine are readily obtained
by the applying the material on Silica GF and
developing with
TOLUNE : ETHANOL : conc NH4OH U.V. (254 & 365
nm)
( 34 : 15 : 1 ) Rf value 0.35
IODINE VAPOUR Rf value 0.50

15. (2) HIGH PERFORMANCE LIQUID CHROMATGROPHY:
 High Performance Liquid Chromatography (HPLC) is a form of
column chromatography that pumps a sample mixture or
analyte in a solvent (known as the mobile phase) at high
pressure through a column with chromatographic packing
material (stationary phase)
 For mefloquine the HPLC method is useful for the qualitative estimation.
 COLUMN : Silica column
 MOBILE PHASE: Methanol : Hexane : conc NH4OH
 DETECTOR : U.V. detection at 280nm
 FLOW RATE : 1.5 ml/ min

16. (3) U.V. SPECTROSCOPY METHOD:
As using the solution
in 0.1N HCl the Mefloquine
shows the λ max at
317 nm
283 nm
222 nm

17. 3.PYRIMETHAMINE
Molecular formula:-C12H13ClN4
Molecular weight:-248.71
Color and odor:-White in color and odorless
Solubility:-practically insoluble in water, soluble in ethanol.

18. Methods of Analysis
Identification tests
Gravimetric method
Non – aqueous titration
Paper chromatographic method
U.V. spectroscopy

19. Identification Tests
Dissolve 0.05 of drug in 5 ml dilute Sulphuric acid and 0.2
ml of alkaline potassium mercuric iodide a creamy white
precipitate is formed
Ignite 0.1 gm with 0.5gm of anhydrous sodium carbonate
extract the residue with water, and filter. The filtrate after
neutralization with nitric acid, yields the reaction
characteristics of Chlorides

20. Gravimetric method
In this method, pyrimethemine is determined
gravimetricaly by precipitation from 5% H2S04
solution with phosphotungestic acid ,washing with
2% H2S04 and drying at 500c for 2Hrs over P2O5 in
vacuum for less then 4 Hrs then heating at 1100c for
1.5Hrs. Each gram is equivalent to 0.204gm of
C12H13N4Cl.

21. Non – aqueous titration
In this method the pyrimethamine is analyzed
by titrating against the 0.1N acetous perchloric acid
using quinalidine red as an indicator. The end point
is pink colour

22. Paper Chromatography
Method I
In this method solvent system used is citric acid:water:n-
butanol(4.8:127:870ml) and the visualizing agent used is UV or
iodoplatinate spray the Rf value found to be 0.42
Method II
In this method solvent system used is acetate buffer (pH 4.58 )
and visualizing method used is UV and Rf value is 0.42 .

23. Ultraviolet spectrum:-
The UV spectrum of a
solution in 0.005N Hcl
exhibits a maximum at
about 272nm .

24. REFRENCE
Analytical profile of drug substances by KLAUS FLOTEY
Volume – 5 page No: 61 to 87
Volume – 12 464-478, 548-608
Volume - 14 page No: 157 to 180