Development and Validation of a RP HPLC Method for the Determination of Anastrozole in Bulk and Dosage Forms

G. Ram Kumar
Pydah College P.G. Courses Visakhapatnam

Introduction
Anastrozole is chemically described as 1,3Benzenediacetonitrile, ,,1,1 -tetramethyl-5- (1H-1,2,4triazol-1-ylmethyl). Its molecular formula is C17H19N5 and its structure is

Introduction - Method of Action

Anastrozole decrease the amount of estrogen, body products and can slow or stop the growth of many types of breast cancer cells1. It is a non steroidal aromatase inhibitor, which converts androgens to estrogens. Anastrozole is manufactured and marketed as ARIMIDEX by ASTRAZENECA. It is available as 1mg tablet supplied in bottles containing 30 tablets
1. P.V. Plourde, M. Dyroff, M. Dukes, Breast Cancer Res. Treat. 30 (1994)103.

Literature
A liquid chromatography method has been developed for the quantitative determination of anastrozole by Saravanan et.al2 An HPLC MS MS method has been developed for measuring plasma anastrozole to quantify anastrozole in human plasma was developed by Mendes et. Al3 Anastrozole has been determined in human plasma by Capillary Gas Chromatographic assay with 63Ni electron capture detector by 4 ,5, 6
2. G. Saravanan, M. V. Suryanarayana, Manoj J. Jadhav, M. Ravikumar,N. Someswararao, P. V. R. Acharyulu, Chromatographia, 66 (2007), 435-438 3. Mendes GD, Hamamoto D, Ilha J, Albeeto dos Santos Pereira A, De Nucci G J. Chromator. B, 850 (2007), 553-559. 4. M.J. Bock, I. Bara, N. LeDonne, A. Martz, M. Dyroff, J. Chromatogr. B700 (1997) 131. 5. J. Yuan, P.Q. Wang, S.R. Ge, F.R. An, A.G. Shi, J. Chen, J.Y. Liang, ActaPharmacol. Sin. 22 (2001) 573. 6. G. Duan, J. Liang, M. Zuo, Biomed. Chromatogr. 16 (2002) 400

Objective of the present study

A sensitive and selective RP HPLC method for

the determination of anaztrozole in bulk and

dosage forms and its validation is presented.

Experimental
1. Chemicals and Reagents
Anastrozole sample was obtained as a gift sample from Dr Reddys laboratories. HPLC- grade acetonitrile were purchased from Merck India Pvt. Ltd., Mumbai, India was used. Distilled water used was prepared by using Millipore Milli Q plus water-purification system. Commercial anastrozole tablets (Arimidex-ASTRAZENECA) was purchased from local market

2. Instrumentation

Shimadzu Prominence HPLC Tokyo- Japan SPD-20A UV/VIS Detector Column - ODS reverse phase column (2504.6 mm I.D, 5) A 20L Hamilton injection syringe was employed.

3. Chromatographic Conditions
Freshly prepared 50:50 v/v mixture of Acetonitrile and Water was used as the mobile phase The flow rate of mobile phase was maintained at 1 mL/min The column temperature was maintained at 25+10 C The detection was carried out at 215 nm. 20L each of the sample is injected in each run.

4. Preparation of Standard Solution

About 20 mg of anastrozole was weighed accurately, transferred into a 20mL volumetric flask, sonicated for 20 minutes and is made up to 20mL until the final concentration is 1mg/mL. Solutions containing 10 to 60 g/mL of anastrozole were subjected to the proposed HPLC analysis.

5. Estimation of ANASTROZOLE in Tablet dosage form

The commercial anastrozole tablet dosage form (Arimidex) of

ASTRAZENECA was chosen for this purpose Sample stock solution is prepared by taking 20 tablets, triturated well, made into fine powder. Equivalent weight of 10mg sample is taken and made into a solution such that final concentration is 1 mg / mL.

6. Method Validation
Method validation was performed following ICH specifications7,8 for precision, range of linearity, accuracy and robustness.
7. International conference on harmonization (1994) Text on Validation of Analytical Procedures Q2A. 8. International conference on harmonization (1996) Validation of Analytical Procedures Methodology Q2B

Results and Discussion

System Suitability
The system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor 2.0 and theoretical plates >2000. The retention time obtained for anastrozole is 2.586 minutes.

anastrozole

Results and Discussion System suitability

S.No 1. 2. 3. 4. 5. (mm) 6. 7. Tailing Factor Peak asymmetry (T) 0.98 0.93 Parameters Retention time in minutes (t) Column Length in cm. (L) Theoretical Plates (n) Theoretical Plates per meter (N) Ht equivalent to Theoretical Plates (HETP) 0.034 Results 2.586 25 7350 51450

Results and Discussion

Precision
The inter- and intra-day means and relative standard deviation (R.S.D.) were calculated and are found to be precise and are the acceptance criteria

Linearity
Test solutions were prepared within the range from 10 - 60 g/mL and injected. The same retention time was observed in all cases. The peak areas were calculated and a regression curve was plotted between peak areas and concentrations.

Results and Discussion

1200000 1000000

Anastrozole Concentration

Average Area 176911.8

353823.5 520735.3 707647 884558.8 1061471

Peak Areas in mV-Sec

800000

(g/ml) 10
20 30 40 50 60
0 10 20 30 40 50 60 70

600000

400000

200000

concentration in g/mL

Linear regression equation is y = 17719x 2666 The Correlation coefficient is R2 = 0.999

Results and Discussion

Accuracy
About 99.64% of anastrozole could be recovered from pre analyzed samples indicating the high accuracy of the proposed method.

Robustness
Typical variations in liquid chromatography conditions were used to evaluate the robustness of the assay method The factors selected were flow rate and percentage composition of mobile phase. The values were found to be under acceptance criteria.

Results and Discussion

Limit of Detection and Limit of Quantification

The limit of detection(LOD) and Limit of Quantification(LOQ)was calculated by the proposed method was based on the standard Deviation of the responses and the slope of the calibration curve are found to be 0.187 g/mL and 0.617 g/mL.

Conclusions
This method can be used for the routine analysis of bulk drug
samples of anastrozole and in pharmaceutical formulations. The method developed for the quantitative determination of

the anastrozole is precise, accurate and selective.

The method is completely validated and satisfactory results were obtained.

Acknowledgements
Grateful to M/s. Dr Reddys laboratories for providing me a gift sample of Anastrozole.