Presentation 1

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)-7chloroquinoline-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 250×4.6mm

M/S Pharmaceuticals

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 in 1000ml Mobile phase Ratio Buffer) Flow rate Injection Volume Detection

dihydrogen Phosphate Water adjust pH 3.0 with Orthophosphoric acid : 50:50(Acetonitrile: : 102ml/min : 20µl : 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

3.

Limits of Quantitation

4.

Limits of Detection

5.

Linearity

Linear Regression coefficient -0.99

Linear Regression 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%

Should not be any interference due to placebo in sample & std. preparation 0.942%

Not more than 2% Up to 0.2 mcg level Artesunate can be determined

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