Simultaneous Spectrophotometric Estimation of Artesunate andMe�o�uine

A simple, rapid, precise, and accurate UV-visible spectrophotometric method has been developed for the simultaneous determination of Artesunate in combination with Me�oquine. For developing the method, methanol was used as a solvent. Artesunate and Me�oquine showed λλmax at 240 nm and 222 nm, respectively. e proposed method was validated as per ICH guideline. e linearity range of Artesunate and Me�oquine were 10–60 and 20–120 μμg/mL, respectively. 99.91 ± 0.2740 and 99.56± 0.2067 these value represent the percent recovery of Artesunate and Me�oquine respectively e correlation coe�cients of Artesunate and Me�oquine were 0.999, and 0.999, respectively. e relative standard deviation for six replicates was always less than 2%. e statistical analysis proves that the method is suitable for the analysis of Artesunate and Me�oquine as the bulk drugs and in pharmaceutical formulation without any interference from the excipients.


Introduction
A simple, rapid, precise, and accurate UV-visible spectrophotometric method has been developed for the simultaneous determination of Artesunate in combination with Me�oquine.ART and MEF are poorly water soluble drugs; therefore, methanol was used as a solvent as it is completely soluble in it.Methanol did not interfere in the spectroscopic determination of ART and MEF having maximum absorbance at 240 nm and 222 nm, respectively.
Artesunate is ideal for the treatment of severe malaria, including cerebral malaria [1,2].
Standard drug sample of Artesunate and Me�oquine was pursued as a gi sample from Cipla Ltd. and Macleoids Ltd.All chemicals and solvents of AR grade were purchased from �ualigens �ne Chemicals, Mumbai, India.
UV-spectrophotometer UV-1800 (Shimadzu, Japan) with spectral bandwidth of 2 nm and 10 mm matched quartz cells were used for the development analytical method over the range of 200-400 nm.Marketed formulation Falcigo Plus tablet containing ART 100 mg and MEF 200 mg was used as sample, purchased from local market.Calibrated glassware was used throughout the work.Similarly, accurately weighed quantity of about 20 mg of pure drug of MEF was dissolved in methanol and diluted to 100 mL.(Concentration 200 g/mL).

Selection of Analytical Wavelengths.
Appropriate dilutions were prepared for each drug from the standard stock solution and scanned in the spectrum mode from 400 nm to 200 nm.ART and MEF showed absorbance maxima at 240 nm (Figure 2) and at 222 nm (Figure 3), respectively.Figure 4 represents the overlay spectra for ART and MEF.

Selection of Analytical Concentration Ranges.
From the standard stock solution of ART, appropriate aliquots were pipetted out into 10 mL volumetric �asks and dilutions were made with methanol to obtain working standard solutions of concentrations 10-60 g/mL.Absorbance for these solutions were measured at 240 nm (Table 1) and a calibration curve of absorbance against concentration was plotted as shown in (Figure 3).Similarly, a series of standard solutions of concentration 20-120 g/mL were prepared for MEF and their absorbance was measured at 222 nm (Table 1).A standard calibration curve of absorbance against concentration was plotted (Figure 4).Both drugs followed the Beers-Lamberts law in the range of 10-60 g/mL and 20-120 g/mL for ART and MEF, respectively.Table 2 summaries the optical characteristics of both drugs.

Determination of Absorptivity Coefficients at Analytical
Wavelengths.e absorptivity coefficients for the two drugs were determined at both the selected wavelengths.e values obtained as the mean of six independent determinations were used for forming the simultaneous equations.e simultaneous equations formed were as follows: at 222 nm (For Me�oquine) at 240 nm (For Artesunate), where  1 and  2 are the absorbance of sample solution at 222 nm and 240 nm, respectively, and  1 and  2 are the concentrations of Me�oquine and Artesunate, respectively, (in gL −1 ) in the sample solution.
By solving the two simultaneous equations, the concentration of me�oquine ( 1 ) and artesunate ( 2 ) in sample solutions can be obtained.

Analysis Standard
Containing ART and MEF.e method was checked by analyzing a solution containing known concentration of both drugs.e mixed standards in the Beer-Lambert's range for each drug in the ratio of 1 : 2 containing 10, 30, and 60 g/mL of ART and 20, 60, and 120 g/mL of MEF, respectively, were prepared by diluting appropriate volumes of standard stock solutions.e scanning of mixed standard solutions was carried out in the range of 400 nm to 200 nm in spectrum mode (Table 3).e absorbance of mixed standard solutions was measured at 240 nm and 222 nm.e concentrations of ART and MEF present in mixed standards were calculated using (1) and (2) (Table 4).e results obtained were good and hence the method was applied to the marketed tablet formulation.
2.6.Procedure for Analysis of Tablet Formulation.Twenty tablets were weighed accurately; the average weight was determined and then triturated to a �ne powder.A quantity equivalent to 100 mg of ART and 200 mg of MEF was weighed and transferred to a 100 mL volumetric �ask containing 70 mL methanol, and the contents were sonicated for 20 min with methanol to dissolve the active ingredients.Volume was made up to 100 mL with methanol and �ltered through �hatman �lter paper no.41 to give the stock solution containing 1000 g/mL of ART and 2000 g/mL of MEF.Various dilutions of the tablet stock solutions were scanned and the absorbance of these solutions were measured at 240 nm and 222 nm, respectively, and the concentrations of the two drugs in the sample solutions were calculated using (1) and ( 2).e analysis procedure was repeated six times.e results of marketed tablet formulation are given in Table 5.

Recovery Studies.
Recovery studies were carried out at three levels that is, 80, 100, and 120% of the label claim of the Tablet formulation as per ICH guidelines [3,4].
To perform recovery studies at 80% of the test concentration, sample containing 100 mg of ART and 200 mg of MEF was weighed and transferred to a 100 mL volumetric �ask.To it, 80 mg of standard ART and 160 mg of standard MEF was added, the mixture was mixed thoroughly.en 70 mL of methanol was added and the contents were sonicated for 20 min with methanol to dissolve the active ingredients, and the volume was made up to 100 mL with methanol and �ltered through �hatman �lter paper no.41.
Similarly, to perform recovery studies at 100% of the test concentration, tablet powder containing 100 mg of ART and 200 mg of MEF was weighed.To it, 100 mg of standard ART and 200 mg of standard MEF was added and at 120% level, 120 mg of standard ART and 240 mg of standard MEF was added to the tablet powder equivalent to 100 mg of ART and 200 mg of MEF.en 70 mL of methanol was added, the contents were sonicated for 20 min with methanol to dissolve the active ingredients, and the volume was made up to 100 mL with methanol and �ltered through �hatman �lter paper no.41.
From the stock solutions prepared at each level, suitable aliquots were pipetted out and diluted to 10 mL with methanol and were analysed as per the procedure for tablet formulations.e results of the recovery studies were also validated statistically.e results of recovery studies are given in Table 6.

Precision of Method.
Precision of the method was veri�ed by using stock solutions in the ratio of 1 : 2 containing 60 g/mL ART and 120 g/mL of MEF.System repeatability was done by repeating the assay three times of six replicate dilutions of the same concentration aer every two hours on the same day for intraday precision.Interday precision was carried out by performing the assay of six sample sets aer 24 hours and 48 hours.e results of intermediate precision are given in Table 7.

Conclusion
e novel method for simultaneous estimation of ART and MEF was developed using alcoholic solubilization technique.ART and MEF follow Beer-Lambert's law in range Plus samples, respectively, indicating that the method has required accuracy and there was no interference from the common excipients present in tablets.e RSD value below 2% indicated that the method has required precision.LOD and LOQ values at 240 and 222 were found to be 0.54 and 0.45 g/mL and 1.79 and 1.48 g/mL, respectively.us, the developed method was simple, accurate, and precise and can be used for routine analysis of ART and MEF in pharmaceutical preparation.

2. 1 .
Preparation of Standard StockSolutions.An accurately weighed quantity of about 10 mg of pure drug of ART was dissolved in methanol and diluted to 100 mL.(Concentration 100 g/mL).

F 1 :
(a) Structure of Artesunate and (b) structure of Me�oquine.

F 2 :
Overlay spectrum of the ART and MEF.

F 4 :
Calibration curve of MEF.
1: Standard calibration table for ART and MEF.

T 2 :
Optical characteristics and other parameters.
T 3: Absorbance of mixed standards containing ART and MEF.
Absorbance F 3: Calibration curve of ART.
T 7: Results of intermediate precision.and 20-120 g/mL shows ART and MEF can be estimated in Methanol.Commercial formulation containing ART and MEF were analyzed by proposed method Mean assay values in Falcigo Plus were found to be 99.78 ± 0.1754 and 99.69 ± 0.3287, respectively.e accuracy of method was determined by recovery studies.Pure ART and MEF were added to the preanalyzed tablet powder at three different levels, namely, 80, 100, and 120% of labeled claims as per the ICH guidelines.ree replicate analyses were carried out at each level.e mean recovery was found to be 99.91 ± 0.2740% and 99.56 ± 0.2067% in Falcigo