Analytical Method Development and Validation of Anostrozole in Pure and Tablet Dosage Form by UV Spectroscopy

 

K.R Sreejith*, P.L Rajagopal, K Premaletha

Academy of Pharmaceutical Sciences, Pariyaram Medical College, Kannur, Kerala.

 

ABSTRACT:

The present study deals with development and validation of a simple, rapid, accurate, economical and reproducible UV-Spectrophotometric method for estimation of Anostrozole in pure form and in tablet dosage form. Area under the curve in the range of 220-231nm of the absorption spectrum was selected for the analysis. Linearity range was found to be 4-20 μg/ml. The correlation coefficient was found to be 0.9997. The molar absorptivity was found to be 37430 L mol/cm. The limit of detection and limit of quantification were found to be 0.3560and 1.0789 μg /ml respectively.  The assay value of Anostrozole in bulk and formulation was calculated at different time intervals for intraday and interday experiments. The proposed method was successfully applied for the determination of Anostrozole in bulk form and in Pharmaceutical formulation.

 

 

 

INTRODUCTION:

Anastrozole is a nonsteroidal aromatase inhibitor used in the treatment of breast cancer. It decreases the amount of estrogen that body makes. This can slow or stop the growth of many types of breast cancer cells that need estrogen to grow. Chemically it is (2-[3-(2-cyanopropan-2-yl)-5-(1,2,4-triazolylmethyl)phenyl]-2-methyl-propanenitrile) having Molecular Formula- C₁₇H₁₉N₅ and molecular weight 293.4 (Figure 1) . It is a white crystalline solid, odorless and is freely soluble in methanol, acetone, ethanol and tetrahydrofuran, and very soluble in acetonitrile having melting point 81-82°C. Anastrozole acts by inhibiting the enzyme aromatase, which is responsible for converting androgens to estrogen. Absorption of Anastrozole is rapid and maximum plasma concentrations typically occures within 2 hours of dosing under fasted conditions.

 

Orally administered Anastrozole is well absorbed into the systemic circulation. Anastrozole is eliminated slowly with a plasma elimination half-life of approximately 50 hours in postmenopausal women. The protein binding of Anastrozole to plasma proteins is about 40% and independent of concentration over a range which includes therapeutic concentrations.^1-3

 

Figure 1. Anastrozole

 

As per the literature few methods for quantitative determination of Anastrozole were reported^4-13.Most of these methods were applied for the determination of Anastrozole in biological fluids and are mainly useful for therapeutic monitoring of the drug. Very few methods for quantitative determination of Anastrozole in bulk drug samples and formulations were reported. So aim of the present study was to develop a simple, reproducible and accurate Spectrophotometric method for the determination of Anastrozole in dosage form.

 

MATERIALS AND METHODS:

Materials and instruments used:

Materials:-

Only A R grade reagents and solvents were used. Anastrozole was obtained as a gift from Dr Reddy’s Laboratories, Hyderabad, India.Armotraz-1mg (Cipla) was purchased from a local pharmacy. Analytical reagent grade ethanol was used.

 

Standard solution:-

Accurately weighed quantity 50 mg of Anastrozole was dissolved in ethanol and the volume was made up to 100 ml to get a stock solution of  500 μg/ml.

 

Instruments:-

        A double beam UV-VIS spectrophotometer (UV-1700, Shimadzu, Japan) connected to a computer loaded with spectra manager software UV Probe, with 1.0 cm quartz cells, was used. The spectra were obtained with the instrumental parameters as follows: wavelength range: 200 - 300 nm; scan speed: medium; spectral slit width: 1 nm. All weights were taken on an electronic balance (Shimadzu, Japan). Double distilled Water and ethanol were used as a solvent.

 

Method development:

Aim of the present study was to develop a simple, reproducible and accurate Spectrophotometric method for the determination of Anastrozole in dosage form. This method was developed by exploiting the absorption spectrum obtained by dissolving drug in ethanol in the presence of sulphuric acid. The method involves the calculation of integrated value of absorbance with respect to the wavelength between two selected wavelength λ1 and λ 2. Area calculation processing item calculates the area bound by the curve and the horizontal axis. The horizontal axis is selected by entering the wavelength range over which the area has to be calculated. The wavelength range is selected on the basis of repeated observations so as to get the linearity between area under curve and concentration. From the spectra of drug, area under the curve in the range of 220-231nm was selected for the analysis.

 

Construction of calibration Curve:

The calibration curve was prepared in the concentration range of 4-20μg/ml at their respective AUC range as follows. 2ml of the stock solution was taken in a 25 ml standard flask and added 1 ml of concentrated sulphuric acid and heated gently at controlled temperature (70-80°C) for 5 minutes. The solution was cooled to room temperature and made up to the mark with ethanol. From this 1-5 ml were taken in 10ml standard flasks and made up to the mark with distilled water. Absorption spectrum was plotted against the blank prepared under similar conditions. Area under the curve for each absorption spectrum was calculated and plotted on Y axis against concentration on X axis.

 

Analysis of dosage forms by the proposed method:

20 tablets were weighed and average weight was calculated. The tablets were crushed to obtain very fine powder. A portion of the powder equivalent to 1mg of anastrozole   was weighed and added to 20 ml of ethanol and shaken well for 10 minutes and the mixture was sonicated for 25 minutes with intermittent vigorous shaking. The solution was centrifuged at 1000rpm for 20 minutes and to the supernatant solution added 1ml of concentrated sulphuric acid and heat gently at controlled temperature (70-80) for 5 minutes. The solution was cooled to room temperature and made up to 25 ml with ethanol. 3ml of this solution was made up to 10 ml with water and reading was taken against the blank prepared in the similar conditions. From the value obtained amount of drug in the dosage form was determined by applying least square method.

 

Recovery study:

Recovery experiments were performed by applying the standard addition technique. A portion of the powder equivalent to 1mg of Anastrozole  was weighed and added to 20 ml of ethanol, to that added definite amount of  standard solution  and shaken well for 10 minutes and the mixture was sonicated for 25 minutes with intermittent vigorous shaking. The solution was centrifuged at 1000rpm for 20 minutes and to the supernatant solution added 1ml of concentrated sulphuric acid and heat gently at controlled temperature (70-80) for 5 minutes. The solution was cooled to room temperature and made up to 25 ml with ethanol. 2ml of this solution was made up to 10 ml with water and reading was taken against the blank prepared in the similar conditions.

 

Method development and optimization:

From the spectra of drug, area under the curve in the range of 220-231nm was selected for the analysis (Figure2). The calibration curve was prepared in the concentration range of 4-20μg/ml at their respective AUC range. (Figure 3)(Table1). By using the calibration curve, the concentration of the sample solution can be determined.

 

Figure 2. Absorption Spectrum and Area Under the Curve

 

Figure 3. Calibration Curve

 

Table 1. Preparation of calibration curve

 

Method validation.

The proposed method was validated as per the ICH guidelines ¹⁴.Validation parameter performed include linearity, limit of detection/quantitation, selectivity, specificity, ruggedness, accuracy and precision.

 

Linearity.

To study the linearity of the method calibration curve was prepared using five points. A series of drug containing4µg/ml,8µg/ml,12µg/ml, 16µg/ml, and 20µg/ml was prepared and the calibration curve was prepared in the concentration range of 4-20μg/ml. Table 1. Calibration curve was constructed by taking concentration on X axis and AUC on Y axis (Figure3). The data was processed statistically to calculate parameters like coefficient of correlation, linearity range, slope, intercept, etc. Table 2.The coefficient of determination from the linear regression analysis was calculated and found to be 0.9997.This indicates that there exists a good linear relationship between concentration and AUC.

Table 2. Optical characters of the proposed methods

Accuracy:

To assess the accuracy of the method recovery experiments were performed by applying the standard addition technique. Data are given in Table3. The recovery range was found to be 98.39-101.44 %.

 

 

Table 3. Data for Recovery Studies

Regression equation  Y= 1.17645X+ 0.029 where  Y = Mean AUC and X = Concentration

*Mean of three observations

 

Table 4. Assay of Tablets

Regression equation  Y= 1.17645X+ 0.029 where  Y = Mean AUC and X = Concentration

*Mean of three observations

 

Table 5. Data for intraday and interday precision (n-=6)

 

Precision:

The precision of the analytical method is expressed as standard deviation or coefficient of variation. The precision study of the proposed method was done based on the data obtained from Table 4.Results of six determination from same batch were validated statistically and standard deviation and coefficient of variation in% was found to be 0.00283 and 0.2819 respectively. The data for intraday and interday precision is given in Table 5.

 

Ruggedness.

The degree of reproducibility of test results obtained by the methods was checked by analyzing the drug sample

 

by different analysts. To validate and confirm the results, six solutions of drug were prepared and analysis was carried out. The data's given in Table 6.

 

Table 6. Ruggedness of the proposed   method

Parameter	Content of drug*	Std Deviation	%RSD
Analyst 1	1.0018	0.00198	0.1973
Analyst 2	1.0046

* Mean of six observations

 

Application of the method in formulation.

The suggested method was applied successfully to the determination of anastrozole in formulations. Armotraz 1 mg tablets were used for the study. Six replicate determinations were made. Satisfactory results were obtained and were in a good agreement with the label claim. Data given Table4.

 

CONCLUSION:

The method was developed by exploiting the absorption spectrum of a solution of Anastrozole in ethanol in presence of concentrated sulphuric acid. The method was validated for selectivity, linearity, limit of detection, limit of quantitation, ruggedness and accuracy. The selectivity of the method was determined by assessing interference from the excipients. The method was linear over the concentration range of 4-20 μg/ml (r2 = 0.9997) with a limit of detection and quantitation of  0.3560 and 1.0789 μg/ml .  Accuracy was determined by recovery experiments and recovery was between 98.39-101.44 %. The methods were found suitable for the assay of Anastrozole in formulation and can be used for routine quality control analysis.

 

ACKNOWLEDGEMENT:

We wish to thank Dr Reddy’s Laboratories, Hyderabad, India for providing the gift sample of Anastrozole.

 

REFERENCES:

1.        Budavari S, O’Neil M, Ann Smith, Heckelman P , Obenchain J. Anastrozole, The Merck Index An Encyclopedia of Chemicals, Drugs, and Biological, Merck Research Laboratories Division of Merck  and  Co., Inc., Whitehouse Station, NJ,12, 668. 1996

2.        Wellington K, Faulds D M. Drugs, 62(17), 2483-2490. 2002

3.        Plourde P V, Breast Cancer Res Treat, 30, 103-111. 1994

4.        Duan G, Liang J, Zuo M. Rapid determination of Anastrozole in plasma by gas chromatography with electron capture detection and its application to an oral pharmacokinetic study in healthy volunteers. Biomed Chromatogr. 16(6); 2002:400-403.

5.        Saravanan G, Suryanarayana M.V, Manoj Jadhav, M. Ravikumar. The stress stability behavior and development of an LC assay method for Anastrazole. Chromatographia. 6(6); 2007:435-438.

6.        Apostolou Constantinos, Dostilla Yannis, Kousoulos Constantinos, Loukas Yannis. Development and validation of an improved high-throughput method for the determination of Anastrozole in human plasma by LC-MS/MS and atmospheric pressure chemical ionization. Journal of Pharmaceutical and Biomedical Analysis. 4(8); 2008: 853-859.

7.        Arvind G. Jangid, Ashutosh M. Pudage, Santosh S. Joshi, Pramod N. Pabrekar, Rajesh H. Tale, Vikas V. Vaidya. A simple, selective and rapid validated method for estimation of Anastrazole in human plasma by liquid chromatography–tandem mass spectrometry and its application to bioequivalence study. Biomedical Chromatography. 24(7); 2009: 727–731.

8.        Sathis Kumar D, Harani A, Rohit Reddy T, Sucharitha G, Krishna. P, Priyanka Sagar J. Development and validation of Spectrophotometric method for estimation of Anastrozole bulk and pharmaceutical dosage formulation. International Journal of Advances in Pharmaceutical Sciences. 1(1); 2010:329-333.

9.        Perumalla Bharati, Arya Vinodini, Ala Shiva Reddy, Potturi Sita Devi. Development and Validation of a Planar Chromatographic Method with Reflectance Scanning Densitometry for Quantitative Analysis of Anastrozole in the Bulk Material and in Tablet Formulations. Journal of Planar Chromatography 23 (1); 2010: 79–83.

10.     Yu J, He J, Zhang Y, Qin F, Xiong Z, Li F. An ultra performance liquid chromatography-tandem mass spectrometry method for determination of anastrozole in human plasma and its application to a pharmacokinetic study. 25(4); 2011:511-516.

11.     Sathis Kumar D, Harani A, Sridhar D, David Banji, Rao K.N.V, Guruviah  Yogeswaran. Development and Validation of a HPLC Method for determination of Anastrozole in Tablet Dosage Form. E-Journal of Chemistry. 8(2); 2011:794-797.

12.     Krishnaveni K, Nalini Y, Srinivas P. Development and Validation of stability indicating RP-HPLC method for estimation of Anastrozole in Bulk and Pharmaceutical Dosage Form. Int J Pharm Sci. 3(1); 2013:375-80.

13.     Mrityunjay Banerjee, Tejaswini Kumari Dash, Ankita Kumari and Swapneswar Khatua. Optimized UV-Vis spectrophotometric method for estimation of anastrozolein pharmaceutical solid dosage form. Der Pharma Chemica, 6(3); 2014:140-144

14.     Validation of Analytical Procedures. Methodology.ICH Harmonised Tripartite Guidelines, 1-8. 1996.

 

 

 

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