# Simultaneous Spectrophotometric Estimation of Artesunate and Mefloquine

Authors: T. M. Kalyankar; R. B. Kakade; M. S. Attar; A. R. Kamble
Journal: Journal of Chemistry
 (2013)

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## Abstract

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


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## Body

## 1. Introduction

A simple, rapid, precise, and accurate UV-visible spectrophotometric method has been developed for the simultaneous determination of Artesunate in combination with Mefloquine. 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.Chemically Artesunate is (3R,5aS,6R,8aS,9R,10S,12R,12aR)-Decahydro-3,6,9 trimethyl-3,12-epoxy-12H-pyrano [4,3-j]-1,2benzodioxepin-10-ol, hydrogen succinate, while Mefloquine  is DL-erythro-&alpha;-2-piperidyl-2,8-bis(trifluoromethyl)-4quinolinemethanol monohydrochloride; (R*,S*)-(±)-& alpha;-2-piperidinyl-2,8-bis(trifluoromethyl)-4-quinolinemethanol monohydrochloride. Structures of ART and MEF are shown in Figure1. Artesunate and its active metabolite dihydroartemisinin are potent blood schizonticides, active against the ring stage of the parasite. Artesunate is ideal for the treatment of severe malaria, including cerebral malaria [1, 2].(a) Structure of Artesunate and (b) structure of Mefloquine.
(a)
(b)Standard drug sample of Artesunate and Mefloquine was pursued as a gift sample from Cipla Ltd. and Macleoids Ltd. All chemicals and solvents of AR grade were purchased from Qualigens fine 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 formulationFalcigo 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.


## 2. Experimental

### 2.1. Preparation of Standard Stock Solutions

An accurately weighed quantity of about 10 mg of pure drug of ART was dissolved in methanol and diluted to 100 mL. (Concentration 100μg/mL).Similarly, accurately weighed quantity of about 20 mg of pure drug of MEF was dissolved in methanol and diluted to 100 mL. (Concentration 200μg/mL).


### 2.2. 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 (Figure2) and at 222 nm (Figure 3), respectively. Figure 4 represents the overlay spectra for ART and MEF.Figure 2
Overlay spectrum of the ART and MEF.Figure 3
Calibration curve of ART.Figure 4
Calibration curve of MEF.


### 2.3. Selection of Analytical Concentration Ranges

From the standard stock solution of ART, appropriate aliquots were pipetted out into 10 mL volumetric flasks and dilutions were made with methanol to obtain working standard solutions of concentrations 10–60μg/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).Table 1
Standard calibration table for ART and MEF.
Sr. no.
For Artesunate
For Mefloquine
Conc. (μg/mL)
Abs.* at 240 nm
Conc. (μg/mL)
Abs.* at 222 nm
(
1
)
10
0.037
20
0.132
(
2
)
20
0.07
40
0.28
(3)
30
0.107
60
0.397
(4)
40
0.139
80
0.548
(5)
50
0.175
100
0.676
(6)
60
0.208
120
0.796
*Each value is a mean of six observations.Similarly, a series of standard solutions of concentration 20–120μg/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 μg/mL and 20–120 μg/mL for ART and MEF, respectively. Table 2 summaries the optical characteristics of both drugs.Table 2
Optical characteristics and other parameters.
Parameters
ART
MEF
Working wavelength (nm)
240
222
Linearity range (μg/mL)
10–60
20–120
Molar absorptivity
3.7
6.6
Limit of detection (μg/mL)
0.54
0.45
Limit of quantitation (μg/mL)Y=mx+c
1.79
1.48
Slope
0.003
0.006
Intercept
0.0001
0.0003
Regression coefficient
0.999
0.998


### 2.4. Determination of Absorptivity Coefficients at Analytical Wavelengths

The absorptivity coefficients for the two drugs were determined at both the selected wavelengths. The values obtained as the mean of six independent determinations were used for forming the simultaneous equations.The simultaneous equations formed were as follows:(1)A1=6.95×C1+4.2×C2
at 222 nm (for Mefloquine)
(2)A2=3.5×C1+6.58×C2
at 240 nm (for Artesunate), where A1 and A2 are the absorbance of sample solution at 222 nm and 240 nm, respectively, and C1 and C2 are the concentrations of Mefloquine and Artesunate, respectively, (in gL−1) in the sample solution. By solving the two simultaneous equations, the concentration of mefloquine (C1) and artesunate (C2) in sample solutions can be obtained.


### 2.5. Analysis Standard Containing ART and MEF

The method was checked by analyzing a solution containing known concentration of both drugs. The mixed standards in the Beer-Lambert’s range for each drug in the ratio of 1 : 2 containing 10,30,and 60μg/mLofART and20,60,and120 μg/mL of MEF, respectively, were prepared by diluting appropriate volumes of standard stock solutions. The scanning of mixed standard solutions was carried out in the range of 400 nm to 200 nm in spectrum mode (Table 3). The absorbance of mixed standard solutions was measured at 240 nm and 222 nm. The concentrations of ART and MEF present in mixed standards were calculated using (1) and (2) (Table 4). The results obtained were good and hence the method was applied to the marketed tablet formulation.Table 3
Absorbance of mixed standards containing ART and MEF.
Sr. no.
Mixed Standards
Abs. at 240 nm
Abs. at 222 nm
Conc. of ART (μg/mL)
Conc. of MEF (μg/mL)
(
1
)
10
20
0.039
0.134
(
2
)
30
60
0.112
0.399
(
3
)
60
120
0.204
0.790Table 4
Results of mixture containing ART and MEF.
Sr. no.
Amount present* (μg/mL)
Amount found* (μg/mL)
% Amount found*
ART
MEF
ART
MEF
ART
MEF
(
1
)
10
20
9.98
19.89
99.80
99.45
(
2
)
30
60
29.85
59.76
99.50
99.60
(
3
)
60
120
59.93
119.65
99.88
99.70
*Each value is a mean of six observations.


### 2.6. Procedure for Analysis of Tablet Formulation

Twenty tablets were weighed accurately; the average weight was determined and then triturated to a fine powder. A quantity equivalent to 100 mg of ART and 200 mg of MEF was weighed and transferred to a 100 mL volumetric flask 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 filtered through Whatman filter paper no. 41 to give the stock solution containing 1000μg/mL of ART and 2000 μg/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). The analysis procedure was repeated six times. The results of marketed tablet formulation are given in Table 5.Table 5
Results of marketed tablet formulation.
Sr. no.
Label claim (mg/tab)
Amount found (mg/tab)
% of Label claim
ART
MEF
ART
MEF
ART
MEF
(
1
)
100
200
99.63
198.72
99.63
99.36
(
2
)
100
200
99.70
199.44
99.70
99.72
(
3
)
100
200
100.02
198.88
100.02
99.44
(
4
)
100
200
99.91
199.96
99.91
99.98
(
5
)
100
200
99.86
198.96
99.86
99.48
(
6
)
100
200
99.57
200.36
99.57
100.18
Mean
99.78
99.69
SD
0.1754
0.3287
% RSD
0.1757
0.3297
Formulation:Falcigo Plus(Zydus Cadila  Pharmaceutical Ltd., Ahmadabad).


### 2.7. 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 flask. To it, 80 mg of standard ART and 160 mg of standard MEF was added, the mixture was mixed thoroughly. Then 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 filtered through Whatman filter 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. Then 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 filtered through Whatman filter 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. The results of the recovery studies were also validated statistically. The results of recovery studies are given in Table6.Table 6
Results of recovery studies.
Level of (%) recovery
Amount present(mg/tab)
Amount of standard added (mg)
Total amountrecovered(mg)
% Recovery*
ART
MEF
ART
MEF
ART
MEF
ART
MEF
80
100
200
80
160
179.28
359.24
99.59
99.79
100
100
200
100
200
199.8
398.46
99.89
99.61
120
100
200
120
240
220.6
436.90
100.26
99.29
% Mean
99.91
99.56
SD
0.2740
0.2067
RSD
0.2742
0.2076
*Each value is the mean of three observations.


### 2.8. Precision of Method

Precision of the method was verified by using stock solutions in the ratio of 1 : 2 containing 60μg/mL ART and 120 μg/mL of MEF. System repeatability was done by repeating the assay three times of six replicate dilutions of the same concentration after every two hours on the same day for intraday precision. Interday precision was carried out by performing the assay of six sample sets after 24 hours and 48 hours. The results of intermediate precision are given in Table 7.Table 7
Results of intermediate precision.
Formulation
Parameter
Intraday precision*
Interday precision*
ART
Mean
99.59
99.69
SD
0.3177
0.3723
% RSD
0.3190
0.3734
MEF
Mean
100.21
99.49
SD
0.5657
0.2502
% RSD
0.5645
0.2515


## 2.1. Preparation of Standard Stock Solutions

An accurately weighed quantity of about 10 mg of pure drug of ART was dissolved in methanol and diluted to 100 mL. (Concentration 100μg/mL).Similarly, accurately weighed quantity of about 20 mg of pure drug of MEF was dissolved in methanol and diluted to 100 mL. (Concentration 200μg/mL).


## 2.2. 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 (Figure2) and at 222 nm (Figure 3), respectively. Figure 4 represents the overlay spectra for ART and MEF.Figure 2
Overlay spectrum of the ART and MEF.Figure 3
Calibration curve of ART.Figure 4
Calibration curve of MEF.


## 2.3. Selection of Analytical Concentration Ranges

From the standard stock solution of ART, appropriate aliquots were pipetted out into 10 mL volumetric flasks and dilutions were made with methanol to obtain working standard solutions of concentrations 10–60μg/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).Table 1
Standard calibration table for ART and MEF.
Sr. no.
For Artesunate
For Mefloquine
Conc. (μg/mL)
Abs.* at 240 nm
Conc. (μg/mL)
Abs.* at 222 nm
(
1
)
10
0.037
20
0.132
(
2
)
20
0.07
40
0.28
(3)
30
0.107
60
0.397
(4)
40
0.139
80
0.548
(5)
50
0.175
100
0.676
(6)
60
0.208
120
0.796
*Each value is a mean of six observations.Similarly, a series of standard solutions of concentration 20–120μg/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 μg/mL and 20–120 μg/mL for ART and MEF, respectively. Table 2 summaries the optical characteristics of both drugs.Table 2
Optical characteristics and other parameters.
Parameters
ART
MEF
Working wavelength (nm)
240
222
Linearity range (μg/mL)
10–60
20–120
Molar absorptivity
3.7
6.6
Limit of detection (μg/mL)
0.54
0.45
Limit of quantitation (μg/mL)Y=mx+c
1.79
1.48
Slope
0.003
0.006
Intercept
0.0001
0.0003
Regression coefficient
0.999
0.998


## 2.4. Determination of Absorptivity Coefficients at Analytical Wavelengths

The absorptivity coefficients for the two drugs were determined at both the selected wavelengths. The values obtained as the mean of six independent determinations were used for forming the simultaneous equations.The simultaneous equations formed were as follows:(1)A1=6.95×C1+4.2×C2
at 222 nm (for Mefloquine)
(2)A2=3.5×C1+6.58×C2
at 240 nm (for Artesunate), where A1 and A2 are the absorbance of sample solution at 222 nm and 240 nm, respectively, and C1 and C2 are the concentrations of Mefloquine and Artesunate, respectively, (in gL−1) in the sample solution. By solving the two simultaneous equations, the concentration of mefloquine (C1) and artesunate (C2) in sample solutions can be obtained.


## 2.5. Analysis Standard Containing ART and MEF

The method was checked by analyzing a solution containing known concentration of both drugs. The mixed standards in the Beer-Lambert’s range for each drug in the ratio of 1 : 2 containing 10,30,and 60μg/mLofART and20,60,and120 μg/mL of MEF, respectively, were prepared by diluting appropriate volumes of standard stock solutions. The scanning of mixed standard solutions was carried out in the range of 400 nm to 200 nm in spectrum mode (Table 3). The absorbance of mixed standard solutions was measured at 240 nm and 222 nm. The concentrations of ART and MEF present in mixed standards were calculated using (1) and (2) (Table 4). The results obtained were good and hence the method was applied to the marketed tablet formulation.Table 3
Absorbance of mixed standards containing ART and MEF.
Sr. no.
Mixed Standards
Abs. at 240 nm
Abs. at 222 nm
Conc. of ART (μg/mL)
Conc. of MEF (μg/mL)
(
1
)
10
20
0.039
0.134
(
2
)
30
60
0.112
0.399
(
3
)
60
120
0.204
0.790Table 4
Results of mixture containing ART and MEF.
Sr. no.
Amount present* (μg/mL)
Amount found* (μg/mL)
% Amount found*
ART
MEF
ART
MEF
ART
MEF
(
1
)
10
20
9.98
19.89
99.80
99.45
(
2
)
30
60
29.85
59.76
99.50
99.60
(
3
)
60
120
59.93
119.65
99.88
99.70
*Each value is a mean of six observations.


## 2.6. Procedure for Analysis of Tablet Formulation

Twenty tablets were weighed accurately; the average weight was determined and then triturated to a fine powder. A quantity equivalent to 100 mg of ART and 200 mg of MEF was weighed and transferred to a 100 mL volumetric flask 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 filtered through Whatman filter paper no. 41 to give the stock solution containing 1000μg/mL of ART and 2000 μg/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). The analysis procedure was repeated six times. The results of marketed tablet formulation are given in Table 5.Table 5
Results of marketed tablet formulation.
Sr. no.
Label claim (mg/tab)
Amount found (mg/tab)
% of Label claim
ART
MEF
ART
MEF
ART
MEF
(
1
)
100
200
99.63
198.72
99.63
99.36
(
2
)
100
200
99.70
199.44
99.70
99.72
(
3
)
100
200
100.02
198.88
100.02
99.44
(
4
)
100
200
99.91
199.96
99.91
99.98
(
5
)
100
200
99.86
198.96
99.86
99.48
(
6
)
100
200
99.57
200.36
99.57
100.18
Mean
99.78
99.69
SD
0.1754
0.3287
% RSD
0.1757
0.3297
Formulation:Falcigo Plus(Zydus Cadila  Pharmaceutical Ltd., Ahmadabad).


## 2.7. 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 flask. To it, 80 mg of standard ART and 160 mg of standard MEF was added, the mixture was mixed thoroughly. Then 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 filtered through Whatman filter 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. Then 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 filtered through Whatman filter 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. The results of the recovery studies were also validated statistically. The results of recovery studies are given in Table6.Table 6
Results of recovery studies.
Level of (%) recovery
Amount present(mg/tab)
Amount of standard added (mg)
Total amountrecovered(mg)
% Recovery*
ART
MEF
ART
MEF
ART
MEF
ART
MEF
80
100
200
80
160
179.28
359.24
99.59
99.79
100
100
200
100
200
199.8
398.46
99.89
99.61
120
100
200
120
240
220.6
436.90
100.26
99.29
% Mean
99.91
99.56
SD
0.2740
0.2067
RSD
0.2742
0.2076
*Each value is the mean of three observations.


## 2.8. Precision of Method

Precision of the method was verified by using stock solutions in the ratio of 1 : 2 containing 60μg/mL ART and 120 μg/mL of MEF. System repeatability was done by repeating the assay three times of six replicate dilutions of the same concentration after every two hours on the same day for intraday precision. Interday precision was carried out by performing the assay of six sample sets after 24 hours and 48 hours. The results of intermediate precision are given in Table 7.Table 7
Results of intermediate precision.
Formulation
Parameter
Intraday precision*
Interday precision*
ART
Mean
99.59
99.69
SD
0.3177
0.3723
% RSD
0.3190
0.3734
MEF
Mean
100.21
99.49
SD
0.5657
0.2502
% RSD
0.5645
0.2515


## 3. Conclusion

The 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 of 10–60μg/mL, and 20–120 μg/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. The 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. Three replicate analyses were carried out at each level. The mean recovery was found to be 99.91±0.2740% and 99.56±0.2067% in Falcigo Plus samples, respectively, indicating that the method has required accuracy and there was no interference from the common excipients present in tablets. The 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 μg/mL and 1.79 and 1.48 μg/mL, respectively.Thus, the developed method was simple, accurate, and precise and can be used for routine analysis of ART and MEF in pharmaceutical preparation.



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*Source: 679857-2012-11-22.xml*