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 techniquesUV – 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.CHROMATOGRAPHY2.1. 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 : C20H22ClN3O

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 : C15H22O5

Molecular weight: 282.34Melting point : 132-135CClinical 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 workMethod 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

[Table 2] Reagents and Chemicals

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

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 analysisStationary Phase : Kromasil C 18 250×406mmMobile Phase : Acetonitrile : BufferBuffer : 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/minInjection Volume : 20µlDetection : 216nmTemperature : 30°C

FIGURE 1: System Suitability for Artesunate (standard)

Figure 2: System Suitability for Amodiaquine (standard 1)

Figure 3: Specificity for Artesunate (sample 1)

Figure 4: Specificity for Amodiaquine (Standard 1)

Figure 5: Limits of Detection for Artesunate (standard 160mg)

Figure 6: Limits of Detection for Amodiaquine [Standard (4mcg/ml)]

Figure 7: Linearity for Artesunate (solution 1)

Figure 8: Linearity for Amodiaquine (solution 1)

Figure 9: Accuracy for Artesunate (Standard)

Figure 10: Accuracy for Amodiaquine (Standard 1)

Figure11: Reproducibility for Artesunate (standard)

Figure 12: Repeatability for Amodiaquine (Sample 2)

RESULTS AND DISCUSSION ArtesunateS.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 Quantitation

0.942% Not more than 2%

4. Limits of Detection

Up to 0.2 mcg level Artesunate can be determined

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 Precision7.1 Reproducibility

0.259% Not more than 2%

7.2 Repeatability 1.156% Not more than 3%

8. Robustness 0.673% Not more than 2%

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 coefficient -0.99

Linear Regression coefficient 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%

8. Robustness8.1 Wave length (340 nm)8.2 Wave length (340 nm)

0.1958 %

0.238 %

The results obtained from the sample preparation shall be calculated and it should be with in the limits of 98% to 102 %.

•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 valuable suggestions.