Analytical Method Validation of

Artesunate and Amodiaquine

Hydrochloride by Using HPLC and UV
 Pharmaceutical analysis may be defined as
the application of analytical procedures used
to determine the purity, safety and quality of
drugs and chemicals. More recently it also
deals with biological samples in support of
biopharmaceutical and pharmacokinetic
studies. Pharmaceutical analysis includes
both qualitative and quantitative analysis.
Qualitative analysis: deals with
identification of the substance.
Quantitative analysis: deals with the
determination of how much of the constituent
is present.
 1.Spectrophotometer techniques
UV – Visible spectroscopy :
It involves the measurement of the amount of ultraviolet
(190-380 nm) or visible (380-800 nm) radiation absorbed by
a substance in a solution

2.CHROMATOGRAPHY
2.1. HIGH PERFORMANCE LIQUID CHROMATOGRAPHY:
• High resolving power
•Speedy separation
•Continuous monitoring of the column effluent
•Accurate quantitative measurement
•Repetitive and reproducible analysis using the same column
•Automation of the analytical procedure and data handling

2.1.1.Reverse – Phase high performance liquid

chromatography
•M/S Pharmaceuticals, Chennai has launched
the production of Amodiaquine tablets and
tablets containing artesunate which are
necessary for the treatment of malarial drugs.

•In the present study Analytical method

validation of HPLC method and UV method are
undertaken to validate this analytical method.
DRUG PROFILE:
AMODIAQUINE HYDROCHLORIDE:

Molecular formula : C20 H22 ClN3O

Molecular weight : 355.1451

Mechanism of action:
Amodiaquine is an antimalarial with schizonticidal activity. It is
effective against the erythrocytic stages of all 4 species of plasmodium. It is as effective as
chloroquine against chloroquine-sensitive strains of Plasmodium falciparum and is also
effective against some chloroquine-resistant strains.

Use : Anti malarial drug

ARTESUNATE :

Molecular formula : C15 H22 O5

Molecular weight : 282.34
Melting point : 132-135°C
Clinical pharmacology :
Artesunate is a potent blood schizonticide agent for P.
falciparum. Artesunate binds tightly to parasitized
erythrocyte membranes. The functional group responsible
for antimalarial activity of artesunate is endoperoxide
bond.
Plan of work
Method Development

Reverse phase HPLC UV-Spectroscopy

 Validation Parameters

System suitability

Specificity

Limits of Quantitation

Limits of Detection

Linearity

Accuracy

Precision

Robustness
A HPLC method for determination of amodiaquine (AQ),
desethylamodiaquine (DAQ), chloroquine (CQ) and
desethylchloroquine (DCQ) in human whole blood, plasma and
urine is reported. 4-(4-Dimethylamino-1-methylbutylamino)-7-
chloroquinoline-internal standard. (O.M.S. Minzi,et.al ).
A reversed-phase HPLC method was developed and validated
for the quantitative determination of amodiaquine (AQ) and its
metabolite desethylamodiaquine (DAQ) in whole blood
collected on filter paper. (M. Ntale et.al,).
A sensitive and selective ion-pair liquid chromatography–
tandem mass spectrometric method (IP-LC–MS/MS) for the
simultaneous determination of amodiaquine (AQ) and its
active metabolite, N-desethylamodiaquine (AQm), in human
blood has been developed and validated.
Pentafluoropropionic acid (PFPA) was applied as ion-pairing
reagent in reversed-phase chromatographic separation
(Xiaoyan Chen,et.al,).
 [Table 1] Instruments required
S.no Name of Instrument Instrument Number

1. HPLC-Shimadzu SHIM/HPLC/033

2. Analytical Balance SHIM/BAL/004

3. Sonicater BAN/SONICED/001

4. pH meter STL/PHM/001

[Table 2] Reagents and Chemicals

S.no Materials/Reagent Source

1. Artesunate Working Standard M/S Pharmaceuticals

2. Placebo granular powder M/S Pharmaceuticals
3. Potassium dihydrogen Phosphate M/S Pharmaceuticals

4. Acetonitrile M/S Pharmaceuticals

5. Purified water/HPLC grade water M/S Pharmaceuticals

6. Column – Kromasil C18 M/S Pharmaceuticals

250×4.6mm
Chromatographic Condition:
Isocratic HPLC system may be used for the
analysis
Stationary Phase : Kromasil C 18
250×406mm
Mobile Phase : Acetonitrile : Buffer
Buffer : 1.36gm of Potassium

dihydrogen Phosphate
in 1000ml
Water adjust pH 3.0 with
Orthophosphoric acid
Mobile phase Ratio : 50:50(Acetonitrile:
Buffer)
Flow rate : 102ml/min
Injection Volume : 20µl
Detection : 216nm
FIGURE 1: Sys
Figure 3: Sp
Figure 5: Limits o
Figure 7: Line
Figure 9: Ac
Figure11: Repr
 RESULTS AND DISCUSSION Artesunate
S.NO PARAMETERS RSD LIMITS
1. System Suitability 0.259% Less than 2%
2. Specificity Should not be any
interference due to placebo
in sample & std. preparation

3. Limits of 0.942% Not more than 2%

Quantitation
4. Limits of Detection Up to 0.2 mcg level
Artesunate can be
determined
5. Linearity Linear Regression Linear Regression
coefficient -0.99 coefficient should be not
less than 0.97
6. Accuracy 0.511% Not more than 2%
7 Precision
7.1 Reproducibility 0.259% Not more than 2%
7.2 Repeatability 1.156% Not more than 3%
S.NO PARAMETERS RSD LIMITS
1. System Suitability 0.357% Not more than 2%
2. Specificity Should not be any
interference due to placebo
in sample and standard
preparation
3. Limits of Quantitation 0.1682 % Not more than 2%

4. Limits of Detection
5. Linearity Linear Regression Linear Regression coefficient
coefficient -0.99 should be not less than 0.97

6. Accuracy 0.458411 % Not more than 2%

7. Precision
7.1 Reproducibility 0.357 % Not more than 2%

7.2 Repeatability 0.2704 % Not more than 2%

•The HPLC method was validated for various parameters as per
ICH guidelines like system suitability, specificity, and limits of
quantitation, limits of detection, accuracy, linearity, precision
and robustness.

•The percentage RSD of the preparation in each study was

calculated.
•Analytical Sciences 2001, Vol.17 Supplement, Basic Education in
Analytical Chemistry.
•Ashutoshkar “Error in pharmaceutical analysis and statistical
validation” In, Pharmaceutical Drug Analysis, New age international
publishers, pp-71-87.
•Beckett A.H. and Stenlake J.B. Practical Pharmaceutical Chemistry 4th
edition, CBS publishers and distributors, 1997.pp 162-164,275-305.
•Choon-Sheen Lai , N.K. Nair , A. Muniandya, S.M. Mansora, P.L.
Olliarob, V. Navaratnama, “Validation of high performance liquid
chromatography–electrochemical detection methods with simultaneous
extraction procedure for the determination of artesunate,
dihydroartemisinin, amodiaquine and desethylamodiaquine in human
plasma for application in clinical pharmacological studies of artesunate–
amodiaquine drug combination”. Journal of chromatography B, 877
(2009) 558–562.
•I also bestow my genuine thanks to my honorable
institutional Guide Mr. D. RAGHUPRATAP, Professor in
Dept. of Pharmacy, Annamalai University, for his valuable
ideas, guidance and encouragement given to me in
bringing out this work successfully.
•I wish to express my heart-felt gratitude and sincere
thanks to Mrs.V.USHA, Quality Controle Manager, Madras
Pharmaceuticals, for her valuable guidance and supported
for my work on this topic
•I wish to express my heart-felt gratitude to DR.
R.MANAVALAN, Professor & Head, Dept. of Pharmacy,
Annamalai University for constant encouragement
throughout the entire period, which helped me in successful
completion of this work
•I thank the staff of Madras Pharmaceuticals and
Department of Pharmacy, Annamalai University for their