A validated method for development of atovaquone as API and tablet dosage forms by UV spectroscopy.

A simple new spectrophotometric method has been developed for estimation of Atovaquone in bulk and tablet dosage form. Atovaquone is estimated to be 251 nm in methanol. The Beer's law is obeyed in the concentration range of 1-10 μg/mL of the drug. The slope and intercept values are 0.111 and 0.012, respectively. Results of analysis of this method have been validated statically and by recovery studies. The method is applied to the marketed tablet formulation. A result of the analysis of tablet formulation, given as a percentage of label claim ± standard deviation, is 99.14 ± 0.66. The precision and accuracy has been examined by performing recovery studies and found to be 100.09 ± 1.14. The developed method is simple, sensitive, and reproducible, and can be used for the routine analysis of Atovaquone in bulk and tablet dosage form.

INTRODUCTION

Atovaquone [Figure 1] is a potent hydroxyl naphthoquinone with approved use in the USA, Canada and several European countries for the treatment of Pneumocystis carinii pneumonia[1-3] in acquired immunodeficiency syndrome (AIDS) patients intolerant to trimethoprim/sulfamethoxazole. Its potent antiprotozoal activity against Plasmodium, Pneumocystis and Toxoplasma[4-6] had prompted further investigations including clinical trials for treatment of T. gondi encephalitis in AIDS patients.[7] A previous study of atovaquone disposition in humans yielded no evidence of metabolites.[5] To date, the assays published for atovaquone are limited to complex gas chromatographic methods and high-performance liquid chromatography (HPLC) methods with multiple sample preparation and extraction procedures.[8-14]

Figure 1

Chemical structure of atovaquone trans-2-[4-(4-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone

MATERIALS AND METHODS

Apparatus

Shimadzu 1800 double beam spectrophotometer with Shimadzu UV PC software was used for all the spectrophotometric measurements and treatment of data. Zero-order absorption spectra were traced in 1 cm quartz cells over the range of 200–400 nm. Satodius balance with having 0.1 mg sensitivity was used for weighing the samples. Class‘A’ volumetric glass wares were used.

Materials and reagents

Atovaquone was gift sample from Glen Mark Pharmaceutical Ltd., Mumbai and used without further purification. Methanol AR Grade was procured from Rankem Chemicals. All the solvents used in spectrophotometric analysis were of analytical reagent grade.

Procedure

Preparation of standard stock solution

About 10 mg of the drug was accurately weighed and transferred to a 100 mL volumetric flask and dissolved in about 25 mL of methanol. The volume was then made up to the mark with methanol. Ten milliliters of this drug solution was transferred to a 100 mL volumetric flask and further diluted up to the mark with methanol. This solution contained 10 μg of drug per milliliter of the solution.

Determination of wavelength of maximum absorbance

Five milliliters of stock solution of Atovaquone was transferred to a 10 ml volumetric flask. It was diluted up to the mark with methanol. The absorbance of the final solution was scanned in the range of 200–400 nm, against methanol as the blank. Atovaquone showed absorbance maxima at 251 nm [Figure 2]. The drug followed linearity in the concentration range of 1–10 μg/mL (Y = 0.111 x + 0.012, R2 = 0.9990) [Figure 3].

Figure 2

UV-spectrum of atovaquone

Figure 3

Calibration curve of atovaquone at 251 nm

Preparation of calibration curve for Atovaquone

Stock solutions of atovaquone (1–10 ml) were pipetted out in to a series of 10 volumetric flask of 10 ml. The volume in each volumetric flask was made up to the mark with methanol and the mixer was shacked. That produced the concentration range of 1–10 μg/ml of Atovaquone. The absorbances of solutions were measured at 251 nm against methanol as blank [Figure 2].

RESULTS AND DISCUSSION

Linearity

Under the experimental conditions described, the graph obtained for UV spectroscopy showed linear relationship. Regression analysis using the method of least-squares was made for the slope, intercept and correlation coefficient values. The regression equations of calibration curves was Y = 0.111 x + 0.012 (R2 = 0.9990) for the UV spectroscopy. The range was found to be 1–10 μg/ml for UV spectrophotometric methods. The statistical parameters given are the regression equation calculated from the calibration graphs, along with the standard deviations of the slope (Sb) and intercept (Sa) on the ordinate. The results are presented in Table 1.

Table 1

Optical characteristics, regression equation and coefficient of the method

Recovery studies and validation of the method according to International conference on Harmonization Guidelines[11–14]

To study the accuracy of the above proposed method, recovery studies were carried out by the addition of the standard drug solution to the placebo, and recovery of the drug was calculated. The result of the recovery studies are summarized in Table 2. The precision of the method was studied by carrying out interday and intraday analysis and was expressed as a relative standard deviation. Specificity was checked by spiking the references standard by placebo. The results were found to be satisfactory and are reported in Table 2.

Table 2

Recovery method from placebo solution

Estimation of Atovaquone in tablet dosage form

For analysis of commercial formulation 20 tablets were weighed accurately and triturated to a fine powder. Powder equivalent to 10 mg of Atovaquone was weighed and transferred to a 100 mL volumetric flask. To this, 25 mL of methanol was added and shaken manually for 15 minutes. The volume was made up to the mark with the same solvent and filtered through Whatmann filter paper No. 42. Ten milliliters of this solution was transferred to a 100 mL volumetric flask for further dilution and contained 10 μg/mL of the solution. An appropriate aliquot was transferred to a 10 mL volumetric flask. The volume was adjusted to the mark and absorbance was recorded at 251 nm. The results were found to be satisfactory and are reported in Table 3.

Table 3

Results of estimation of atovaquone (Mepron)

CONCLUSION

The method for the estimation of Atovaquone in the tablet dosage form was developed. The drug shows absorption maxima at 251 nm. Spectrophotometric method linear response was obtained in the concentration range of 1–10 μg/mL, with a correlation coefficient of 0.9990. The method was statistically validated according to the ICH guidelines. The developed validated method was simple, rapid, precise and accurate. The newly developed method could be used for routine analysis of Atovaquone in tablet dosage forms.