Dev. and Verification of HPCL

7/31/2019 Dev. and Verification of HPCL

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Development and Validation of a RP

HPLCMethod for the Determination of Anastrozole in

Bulk and Dosage Forms

G. Ram Kumar

Pydah College P.G. CoursesVisakhapatnam


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IntroductionAnastrozole is chemically described as 1,3-

Benzenediacetonitrile,,,1,1 -tetramethyl-5- (1H-1,2,4-triazol-1-ylmethyl). Its molecular formula is C17H19N5 and its

structure is


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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 convertsandrogens 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.


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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 formeasuring 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 63Nielectroncapture 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


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A sensitive and selective RP

HPLC method for

the determination of anaztrozole in bulk and

dosage forms and its validation is presented.

Objective of the present study


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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


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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.


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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.


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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.


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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 finalconcentration is 1 mg / mL.

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

6. Method Validation

Method validation was performed following ICH specifications7,8

for precision, range of linearity, accuracy and robustness.


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System Suitability

The system suitability method acceptance criteria set in eachvalidation run were: capacity factor >2.0, tailing factor 2.0

and theoretical plates >2000.

The retention time obtained for anastrozole is 2.586 minutes.

Results and Discussion

anastrozole


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S.No Parameters Results

1. Retention time in minutes (t) 2.586

2. Column Length in cm. (L) 25

3. Theoretical Plates (n) 7350

4. Theoretical Plates per meter (N) 51450

5.Ht equivalent to Theoretical Plates (HETP)

(mm)

0.034

6. Tailing Factor 0.98

7. Peak asymmetry (T) 0.93

System suitabilityResults and Discussion


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Precision

The inter- and intra-day means and relative standarddeviation (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.



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concentration in g/mL

0 10 20 30 40 50 60 70

PeakAreasinmV-Sec

0

200000

400000

600000

800000

1000000

1200000Anastrozole

Concentration Average

(g/ml) Area10 176911.8

20 353823.5

30 520735.3

40 707647

50 884558.860 1061471

Linear regression equation is y = 17719x

2666

The Correlation coefficient is R2 = 0.999



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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.



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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.



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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.

Conclusions


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Acknowledgements

Grateful to M/s. Dr Reddys laboratories for providing me a gift

sample of Anastrozole.


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Dev. and Verification of HPCL

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