Seminar on methods of analysis of antimalarials

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).

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

1.CHLOROQUINE PHOSPHATE

SOLUBILITY : Soluble in WATER, Insoluble in ALCOHOL and CHLOROFORMMOL. WT : 515.87PROPERTIES : White, crystalline powder having bitter test

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

(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.

SYSTEM SPOTTING SOLUTION

DETECTION Rf VALUE

Methanol :Water : Con.

Ammonia (72:25:3)

Chloroform U.V.-254Iodine vapour

.28

Chloroform : Cyclo

Hexane : Diethylamine

(5:4:1)

Water U.V.-254Dragnadroff

reagent

.40

N- Butanol : Con. Ammonia :

Alchol (5:3:2)

Water U.V.-254Dragnadroff

reagent

.60

(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

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.

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

(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

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

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

(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

(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

RETATION TIME : 6 MIN

(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

3.PYRIMETHAMINE

Molecular formula:-C12H13ClN4Molecular weight:-248.71Color and odor:-White in color and odorlessSolubility:-practically insoluble in water, soluble in ethanol.

Methods of Analysis

Identification testsGravimetric method Non – aqueous titrationPaper chromatographic method U.V. spectroscopy

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

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.

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

Paper Chromatography

Method IIn 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 IIIn this method solvent system used is acetate buffer (pH 4.58 ) and visualizing method used is UV and Rf value is 0.42 .

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

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