Spectrophotometric Methods for Estimation of Artesunate from Tablet Dosage forms by Hydrotrophy

 

Anekar V. P.1*, Mohite P. M.1, Sankpal P. S.2

1Govt. College of Pharmacy, Karad (M.S.) India

2 Vasantidevi Patil Institute College of Pharmacy, Kodoli, Kolhapur (M.S.) India.

*Corresponding Author E-mail: vaishnavianekar8@gmail.com

 

ABSTRACT:

Quantitative estimation of poorly water-soluble drugs involves use of organic solvents. Major drawbacks of organic solvents include high cost, volatility and toxicity. In the present investigation, hydrotropic solubilization is employed to enhance the aqueous solubilities of poorly water-soluble drugs like artesunate in tablet dosage forms. This method utilizes 2.0 M urea solution as, hydrotropic solubilizing agent. In the urea solution artesunate shows maximum absorbance at 242nm. The 2.0M urea solution does not show any interference with the sampling wavelength. The hydrotropic agent and additives used in the manufacture of tablets did not interfere in the analysis. The results of tablet analysis were found be in range of 98.56 to 101.02% by hydrotropy. The results of analysis of both methods were validated statistically following ICH guidelines.

 

KEYWORDS: Artesunate, Urea, Hydrotropy.

 

 


INTRODUCTION:

The Artesunate (ART) used as a frontline antimalarial drug recommended by the world health organization for use in the treatment of all forms of malaria affecting humans. [1] The drug has the fastest rate of plasmodium elimination and relieves of malaria symptoms [2]. In recent time there has been a report of treatment failure while using these drugs in the treatment of malaria. [3] Most of these treatment failures are attributed to the use of fake and substandard drugs [4].

 

Artesunate is an anti malarial agent act by cleavage of the peroxide bond through reaction with haeme. This produces free radicals which alkylate parasitic proteins. Slightly soluble in water, acetone, ethanol, methanol and acetonitrile. It has been shown to inhibit an essential parasite calcium adenosine triphosphatase enzyme. [5,6]

 

In the analysis of artesunate, major problem is solubilization of artesunate in most of solvents during analysis. Quantitative estimation of poorly water-soluble drugs involves use of organic solvents. Major drawbacks of organic solvents include high cost, volatility and toxicity. In the present investigation, hydrotropic solubilization is employed to enhance the aqueous solubilities of poorly water-soluble drugs like artesunate tablet dosage forms.

 

Literature survey reveals few analytical methods for estimation of artesunate alone or in combination with other drugs in biological fluids and pharmaceutical formulations which includes HPLC with electrochemical detection and with ion pairing, reversed phase HPLC and colorimetric method [7-14].

 

In a proposed method this problem has been solved by using 2M urea solution. Highly sensitive and economic method is not reported for determination of artesunate. Hence in this communication we have reported new UV spectrophotometric methods for determination artesunate using hydrotropy method.

 

EXPERIMENTAL:

MATERIALS:

Instrument:

Spectrophotometric analysis was carried out on a JASCO UV-spectrophotometer 530 using a 1cm quartz cell. The instrument settings were zero order derivative mode and band width of 2.0nm in the range of 200–400 nm.

 

Reagents and Chemicals:

Artesunate were obtained as a gift sample from Skymax Laboratories Pvt. Ltd., Gujarat. All chemicals were analytical grade obtained from SD fine chemicals. Water purified by glass distillation apparatus.

 

Preparations of Standard Drug Solutions and Reagents:

For hydrotropic solubilization 20mg of pure artesunate was dissolved in 50ml of 2.0M urea solution and stirred for 15 minutes and the final volume of both solutions was made up to 100ml with distilled water. The solution was filtered through Whatmann filter paper No. 41. This solution was further diluted with distilled water to prepare working concentrations of 100μg/ml of artesunate.

 

METHODS:

Preliminary Solubility Studies of Drug:

Solubility of artesunate determined at 28 ± 1°. An excess amount of drug was added to 2M urea solution in vials. The vials were shaken mechanically for 12 h at 28 ± 1°, in a mechanical shaker. These solutions were allowed to equilibrate for the next 24 hours, and then centrifuged for 5 minutes at 2000rpm. The supernatant of each vial was filtered through Whatmann filter paper No. 41. The filtrates were diluted suitably, and analyzed spectrophotometrically against corresponding solvent blank.

 

Spectroscopic method employing hydrotropic solubilization:

From the overlain spectra of the drug artesunate and 2.0 M urea (Figure 1), it was found that the urea used does not interfere with the sampling wavelength. Therefore 2.0M urea is used for the solubilization of drug. Different aliquots of the standard solutions were taken in series of volumetric flasks to prepare the concentrations ranging from 5-30μg/ml and volume was made up to mark with water. These concentrations were scanned in the range 200-400nm range absorbances of each concentration taken at 242nm. Calibration curve was prepared by plotting absorbance against concentration.

 

Figure 1: Overlain Spectra of 5 µg/ml of Artesunate in Urea and 2.0 M Urea Solution.

 

Table 1: Absorbance values for calibration curve of ART at 242 nm

Sr. No.

Concentration (µg/ml)

Absorbance

1.

5

0.2585

2.

10

0.4003

3.

15

0.5557

4.

20

0.6087

5.

25

0.76423

6.

30

0.8154

 

Procedure for analysis of tablet formulation:

Twenty tablets were weighed and ground to a fine powder. Tablet powder equivalent to 30mg artesunate was weighed and transferred to a 100ml volumetric flask 70ml of 2.0M urea solution was added to the flask and stirred for 15 min to dissolve the drug and the final volume was made up to 100ml with distilled water. The solution was filtered through Whatmann fitter paper No. 41 and the first few ml were rejected. The filtrate was diluted suitably with distilled water to get 10μg/ml of artesunate. The absorbance at 242nm was measured and the amount of drug present in the sample solution was obtained from the slope and intercept values obtained from the calibration curve (Table 1). From these, concentrations, the composition of the tablet was obtained. The results of analysis of tablet formulations are recorded in Table 2. After 48 hours, the solutions were reanalyzed to determine chemical stability and precipitation, if any.

 

Table 2. Results of tablet analysis:

Analyte

Label claim

(mg/tab)

% Label claim estimated*

(Mean ± S. D.)

R.S.D.

ART

100

99.59 ± 1.0652

1.0359

* Average of nine determinations; S.D.: standard deviation

 

METHOD VALIDATION:

The method was validated according to ICH Q2B R1 guidelines for validation of analytical procedures in order to determine the linearity, sensitivity, precision, robustness and accuracy for the analyte (15). Accuracy and specificity of analysis was determined by performing recovery studies by spiking different concentrations of pure drug in the preanalyzed tablet sample. Results of validation parameters are reported in Table 3 to 7.

 

Table 3: Results of recovery study:

Analyte

Label claim

(mg)

%Label claim estimated*

(Mean ± S. D.)

R.S.D.

ART

100

100.48 ± 1.256

1.14

* Average of nine determinations; S.D.: standard deviation

 

Table: 4 Results of repeatability:

Analyte

Label claim

(mg)

%Label claim estimated*

(Mean ± S. D.)

R.S.D.

 ART

100

99.64 ± 1.7433

1.752

* Average of nine determinations; S.D.: standard deviation.

 

Table: 5 Results of Intraday Precision:

Time

% Label claim estimated* (Mean ± S.D.)

R.S.D.

ART

ART

T-1

99.29 ± 0.4674

1.1258

T-2

101.04 ± 1.230

1.0236

T-3

99.53 ± 1.2093

1.8952

* Average of nine determinations; S.D: standard deviation.

 

Table: 6 Limit of Detection and Limit of Quantitation:

LOD (µg ml - 1) *

LOQ (µg ml - 1) *

0.056

0.068

* Average of six determinations; R.S.D., relative Standard Deviation.

 

Table: 7 Results of robustness (using ethanol solution):

Analyte

Label claim

(mg)

% Label claim estimated*

(Mean ± S.D.)

R.S.D.

 ART

 100

 99.40 ± 1.231

1.89

*Average of nine determinations; R.S.D., relative Standard Deviation.

 

RESULTS:

Quantitative estimation of poorly water-soluble drugs involves use of organic solvents. Major drawbacks of organic solvents include high cost, volatility and toxicity. In the present investigation, hydrotropic solubilization is employed to enhance the aqueous solubilities of poorly water-soluble drugs like artesunate in tablet dosage forms. The results of solubility studies indicated that enhancement in aqueous solubility of artesunate in 2.0M urea solution was more than 6 to7 folds as compared to their solubilities in distilled water. Therefore, this solution was employed to extract artesunate from the fine powder of tablet formulation and thus analysis will become easier one.

 

The result of analysis of tablet formulation by both methods showed % relative standard deviation values in the range of 0.5859 to 1.039 for ATR, which indicates repeatability of the method. The results indicated excellent recoveries ranging from 98.52 to 101.13% for ATR. Recoveries obtained for the drug do not differ significantly from 100%, which showed that there was no interference from common excipients used in the formulation. The results of limits of detection and quantitation were within limits.

 

DISCUSSION:

It is evident from results of validation studies for both methods that methods are accurate, sensitive, selective, precise and robust for spectroscopic estimation of artesunate. Moreover the method is economic, simple and rapid, hence can be employed for routine analysis in quality control laboratories for estimation of artesunate form marketed formulations. After optimizing these methods to estimate artesunate from biological fluids, these methods can be used in Clinical and Bioequivalence studies.

 

REFERENCES:

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2.      David B, Peter W. Current issues in the treatment of uncomplicated malaria in Africa. British Medical Bulletin. 2004; 71:29-43. 3.

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7.      Nogueira FH, Naialy Fernandes Araújo Reis, “Development and validation of HPLC method for the simultaneous determination of artesunate and mefloquine hydrochloride in fixed-dose combination tablets.” Brazilian Journal of Pharmaceutical Sciences.2013 Vol 49.838-843.

8.      Na-Bungcheng K, Congpuong K, Hung LN, Molunto P, Karborang J. Simple high performance chromatographic method with electrochemical detection for simultaneous determination of artesunate and dihydroartemisinin in biological fluids. Journal of Chromatography. 1998; 708:201-207.

9.      Naik H, Murry DJ, Kirsch LE, Fleckenstein LD. Development and validation of high performance liquid chromatography-mass spectroscopic assay for the determination of artesunate and dihydroartemisinin in human plasma. J Chromatography B. Analy. Technol. Biomed. Life Sci. 2005; 816:233-238

10.    Zhou ZM, Anders JC, Chung H, Theoharides AD. Analysis of artesunic acid and dihydroqinghaosu in blood by high-performance liquid chromatography with reductive electrochemical detection. J Chromatogr 1987; 414:77-90.

11.    Taylor RB, Award MI, Reid RG, Moody RR. Determination of sodium artesunate in plasma using ion pairing high performance liquid chromatography. J Chromatogr B 2000; 744:415-21.

12.    Van Agtmael MA, Butter JJ, Portier EJ, Van Boxtel CJ. Validation of an improved Reversed phase high performance liquid chromatography assay with reductive electrochemical detection for determination of artemisinin derivatives in man. Ther Drug Monit 1998; 20:109-16.

13.    Green MD, Mount DL, Writz RA, White NJ. A colorimetric field method to assess the authenticity of drugs sold as the antimalarial artesunate. J Pharm Biomed Anal 2000; 24:65-70.

14.    Green MD, Mount DL, Writz RA. Authentication of artemether, artesunate and dihydroartemisinin antimalarial tablets using a simple Colorimetric method. Trop Med Int Health 2001; 6:980-2.

15.    International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human use: Harmonised Triplicate Guideline on Validation of Analytical Procedures: Methodology, Recommended for Adoption at step 4 of the ICH Process by ICH steering committee, IFPMA, Switzerland, 1996.

 

 

Received on 12.02.2020         Modified on 27.03.2020

Accepted on 15.04.2020       ©A&V Publications All right reserved

Res.  J. Pharmacology and Pharmacodynamics.2020; 12(2): 88-90.

DOI: 10.5958/2321-5836.2020.00017.8