A New Analytical RP-HPLC Method for the Estimation of Letrozole in Pure and Tablet form

 

V. Ravikumar1*, Chillara Sandhya1, Ramya Sri. S2

1Department of Pharmaceutical analysis, Samskruti College of Pharmacy,

Affiliated to JNTUH University, Hyderabad 501301, Telangana, India.

2Department of Pharmacy, University College of Technology,

Osmania University, Hyderabad, Telangana, 500007, India.

*Corresponding Author E-mail: ravikumarsamskruthi@gmail.com

 

ABSTRACT:

A simple, rapid, specific and accurate reverse phase high performance liquid chromatographic method has been developed for the validated of Letrozole in bulk as well as in marketed pharmaceutical dosage form. This separation was performed on a Symmetry ODS C18 (4.6×250mm, 5µm) column with Methanol: Phosphate Buffer (35:65) V/V as mobile phase at a flow rate of 1.0mL min−1 with UV detection at 240nm; the constant column temperature was Ambient. The runtime under these chromatographic conditions was less than 8 min. The retention time of Letrozole was found to be 2.252. The calibration plot was linear over the concentration range of 6–14μg mL−1 with limits of detection and quantification values of 1.2 and 3.6ng mL−1 respectively. The mean % assay of marketed formulation was found to be 99.86%, and % recovery was observed in the range of 98-102%. Relative standard deviation for the precision study was found <2%. The developed method is simple, precise, specific, accurate and rapid, making it suitable for estimation of Letrozole in bulk and marketed pharmaceutical dosage formdosage form.

 

KEYWORDS: Letrozole, RP-HPLC.

 

 

INTRODUCTION:

HPLC is also being automated which involve automated sampling, separation, detection, recording, calculation and printing of results. HPLC offers a wide choice of chromatographic separation methodologies from normal to reverse phase and whole range of mobile phases using isocratic or gradient elution techniques 1. The packing material of the column is the basic feature for the growth of this technique which directly responsible for the chromatographic separations.

 

The principle of separation of compounds is given by Van Deemter equation, which is an empirical formula that describes the relationship between linear velocity (flow rate) and plate height2.

 

Letrozole 4,4í-(1H-1,2,4-triazol-1- ylmethylene) bisbenzonitrile3, is a potent, specific, non-steroidal, third generation aromatase inhibitor, used therapeutically to treat hormone-sensitive breast cancer in postmenopausal women4.

 

Cancer is a fatal disease. It can be cured if detected in an early stage5. The incidence of breast cancer is rising in every country of the world especially in developing country such as India. There has been no improvement in breast cancer presentation over the past 5–10 years, in spite of breast awareness programmers. Much of the increase of breast cancer in India has been associated with greater urbanization and changing life styles6.

 

Fig. 1: Chemical structure of Letrozole7

 

MATERIALS AND METHODS:

Letrozole (Pure) from Sura labs, Water and Methanol for HPLC from LICHROSOLV (MERCK), Acetonitrile for HPLC from Merck.

 

HPLC Method Development:

Trails:

Preparation of standard solution:

Accurately weigh and transfer 10mg of Letrozole working standard into a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and removal of air completely and make volume up to the mark with the same Methanol.

 

Further pipette 0.1ml of the above Imatinib stock solutions into a 10ml volumetric flask and dilute up to the mark with Methanol.

 

Procedure:

Inject the samples by changing the chromatographic conditions and record the chromatograms, note the conditions of proper peak elution for performing validation parameters as per ICH guidelines.

 

Mobile Phase Optimization:

Initially the mobile phase tried was Methanol and Methanol: Water with varying proportions. Finally, the mobile phase was optimized to Methanol: Phosphate Buffer in proportion 35:65% v/v. 

 

Optimization of Column:

The method was performed with various C18 columns like, X- bridge column, Xterra, and C18 column. Symmetry ODS C18 (4.6 x 250mm, 5mm) was found to be ideal as it gave good peak shape and resolution at 1ml/min flow.

 

Validation methods procedures followed as per ICH guidelines8-11.

RESULTS AND DISCUSSION:

Optimized Chromatogram (Standard):

Mobile phase ratio: Methanol: Phosphate Buffer (35:65) V/V

Column: Symmetry ODS C18 (4.6×250mm, 5µm)

Column temperature: Ambient

Wavelength: 240nm

Flow rate: 1ml/min

Injection volume: 10µl

Run time: 8min

               

Figure-2: Optimized Chromatogram (Standard)

 

Table-1: Optimized Chromatogram (Standard)

S. No.

Name

RT

Area

Height

USP Tailing

USP Plate Count

1

Letrozole

2.252

1658242

185421

1.24

6569

 

Observation:

In this trial it shows proper separation of peak and more plate count in the chromatogram and the tailing factor is within the limit. So it is an optimized chromatogram.

 

Optimized Chromatogram (Sample):

 

Figure-3: Optimized Chromatogram (Sample)

 

 

Table-2: Optimized Chromatogram (Sample)

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

1

Letrozole

2.296

1689654

185231

1.28

6659

 

Table-3 Results of system suitability for Letrozole

S. No

Peak Name

RT

Area (µV*sec)

Height (µV)

USP Plate Count

USP Tailing

1

Letrozole

2.277

1652847

185647

6589

1.24

2

Letrozole

2.277

1653658

186254

6587

1.26

3

Letrozole

2.267

1654521

185475

6584

1.28

4

Letrozole

2.265

1653564

186594

6582

1.29

5

Letrozole

2.277

1658745

185684

6895

1.24

Mean

 

 

1654667

 

 

 

Std. Dev.

 

 

2355.764

 

 

 

% RSD

 

 

0.142371

 

 

 

 

Assay (Standard):

Table-4: Peak results for assay standard

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Letrozole

2.265

1658254

185468

1.24

6391

1

2

Letrozole

2.267

1658475

184524

1.23

6549

2

3

Letrozole

2.267

1658471

186598

1.25

6682

3

 

Assay (Sample):

Table-5: Peak results for Assay sample

S. No

Name

RT

Area

Height

USP Tailing

USP Plate Count

Injection

1

Letrozole

2.246

1645879

184574

0.85

6458

1

2

Letrozole

2.246

1645875

183598

0.86

6584

2

3

Letrozole

2.246

1658423

185472

0.85

6457

3

 

 

% ASSAY =

Sample area        Weight of standard     Dilution of sample    Purity   Weight of tablet

 ___________ ×   ________________ × _______________×_______×______________×100

Standard area      Dilution of standard    Weight of sample      100         Label claim

 

The % purity of Letrozole in pharmaceutical dosage form was found to be 99.86%.

 

Linearity:

Chromatographic Data for Linearity Study:

Table-6: Data for Linearity of Letrozole

Concentration mg/ml

Average Peak Area

6

1078475

8

1461129

10

1808358

12

2211573

14

2593778

 

Fig-4: Linearity Curve of Letrozole

 

Linearity Plot:

The plot of Concentration (x) versus the Average Peak Area (y) data of Letrozole is a straight line.

Y = mx + c

Slope (m) = 18500

Intercept (c) = 16179

Correlation Coefficient (r)   = 0.999

 

Validation Criteria:

The response linearity is verified if the Correlation Coefficient is 0.99 or greater.

 

CONCLUSION:

Correlation Coefficient (r) is 0.99, and the intercept is 0.16179. These   values meet the validation criteria.

 

Precision:

The precision of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed conditions.

 

Repeatability:

 

Table-7: Results of repeatability for Letrozole:

S. No

Peak name

Retention time

Area (µV*sec)

Height (µV)

USP Plate Count

USP Tailing

1

Letrozole

2.293

1658954

186958

1.26

6785

2

Letrozole

2.276

1658745

187548

1.27

6854

3

Letrozole

2.286

1659865

189854

1.26

6852

4

Letrozole

2.277

1653254

186985

1.25

6784

5

Letrozole

2.280

1654781

189542

1.24

6895

Mean

 

 

1657120

 

 

 

Std.dev

 

 

2913.592

 

 

 

%RSD

 

 

0.175823

 

 

 

 

Intermediate precision:

 

Table-8: Results of Intermediate precision Analyst 1 for Letrozole

S. No.

Peak Name

RT

Area (µV*sec)

Height (µV)

USP Plate Count

 

USPTailing

1

Letrozole

2.274

1678541

186589

6587

1.26

2

Letrozole

2.258

1685985

186598

6321

1.26

3

Letrozole

2.267

1685745

186985

6385

1.25

4

Letrozole

2.270

1685987

187854

6580

1.26

5

Letrozole

2.264

1698526

187549

6721

1.27

6

Letrozole

2.265

1685943

186598

6637

1.26

Mean

 

 

1686788

 

 

 

Std. Dev.

 

 

6463.466

 

 

 

% RSD

 

 

0.383182

 

 

 

 

Table-9: Results of Intermediate precision Analyst 2 for Letrozole

S. No

Peak Name

RT

Area (µV*sec)

Height (µV)

USPPlate count

USPTailing

1

Letrozole

2.277

1665847

167481

6854

1.25

2

Letrozole

2.255

1658989

167854

6785

1.26

3

Letrozole

2.265

1659845

167895

6854

1.24

4

Letrozole

2.255

1665964

167854

6895

1.26

5

Letrozole

2.253

1659863

168585

6459

1.25

6

Letrozole

2.252

1665986

167859

6456

1.26

Mean

 

 

1662749

 

 

 

Std. Dev.

 

 

3501.766

 

 

 

% RSD

 

 

0.210601

 

 

 

 

Accuracy:

 

Table-10: The accuracy results for Letrozole

% Concentration

(at specification Level)

Area

Amount Added (ppm)

Amount Found (ppm)

% Recovery

Mean Recovery

50%

109068.3

5

5.021

100.420%

100.72%

100%

202187

10

10.054

100.540%

150%

297032.3

15

15.181

101.206%

 

Limit of Detection For Letrozole:

The    detection  limit  of  an  individual  analytical  procedure  is  the  lowest  amount  of analyte in a sample which can be detected but not necessarily quantitated as an exact value.

 

LOD= 3.3 × σ / s

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

Result:

= 1.2µg/ml

 

Quantitation limit:

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined. 

 

LOQ=10×σ/S

Where 

σ = Standard deviation of the response   

S = Slope of the calibration curve

 

RESULT:

= 3.6µg/ml

 

Robustness:

Table-11: Results for Robustness

Parameter used for sample analysis

Peak Area

Retention Time

Theoretical plates

Tailing factor

Actual Flow rate of 1.0 mL/min

1658242

2.312

6569

1.24

Less Flow rate of 0.9 mL/min

1854215

2.458

6865

1.35

More Flow rate of 1.1 mL/min

1758468

2.032

6254

1.32

 

CONCLUSION:

In the present investigation, a simple, sensitive, precise and accurate RP-HPLC method was developed for the quantitative estimation of Letrozole in bulk drug and pharmaceutical dosage forms.

 

This method was simple, since diluted samples are directly used without any preliminary chemical derivatisation or purification steps.

 

Letrozolewas found to be soluble in organic solvents such as ethanol, DMSO, and dimethyl formamide.

 

Methanol: Phosphate Buffer (35:65) V/V was chosen as the mobile phase. The solvent system used in this method was economical.

 

The %RSD values were within 2 and the method was found to be precise.

 

The results expressed in Tables for RP-HPLC method was promising. The RP-HPLC method is more sensitive, accurate and precise compared to the Spectrophotometric methods.

 

This method can be used for the routine determination of Letrozolein bulk drug and in Pharmaceutical dosage forms.

 

ACKNOWLEDGEMENT:

Thе Authors arе thankful to the Management and Principal, Department of Pharmacy, Samskruti College of Pharmacy, Hyderabad, for extending support to carry out the research work. Finally, the authors express their gratitude to the Sura Pharma Labs, Dilsukhnagar, Hyderabad, for providing research equipment and facilities.

 

REFERENCES:

1.      Govinda Rao Kamala, Sowjanya Vadrevu, Malipeddi Haripriya. Method Development and Validation for Simultaneous Estimation of Omeprazole and Domperidone by RP-HPLC. Asian J. Pharm. Ana. 5(4): 2015; 195-205. doi: 10.5958/2231-5675.2015.00031.9

2.      Hamid Khan, Javed Ali. UHPLC: Applications in Pharmaceutical Analysis. Asian J. Pharm. Ana. 2017; 7(2): 124-131. doi: 10.5958/2231-5675.2017.00020.5

3.      I.E. Otuokere, F. J. Amaku.  Conformational analysis and excited – state properties of a highly potent and totally selective aromatase inhibitor, 4,4-(1H-1,2,4-triazol-1-ylmethanediyl) dibenzo nitrile (letrozole). Research Journal of Pharmacology and Pharmacodynamics. 7(4), 2015, 176-180.

4.      Nita Mondal, Tapan K. Pal, And Saroj K. Ghosal. Development And Validation Of RP-HPLC Method To Determine Letrozole In Different Pharmaceutical Formulations And Its Application To Studies Of Drug Release From Nanoparticles. Acta Poloniae Pharmaceutica ń Drug Research, Vol. 66 No. 1 pp. 11ń17, 2009

5.      Nirmala V, Athahari Begu, Anuradha. Effectiveness of Video Assisted Teaching Programme on Prevention of Oral Cancer among Tobacco Chewers. Asian Journal of Nursing Education and Research., 2012, 2(2), 83-86.

6.      Arjun Patidar, S.C.Shivhare, Umesh Ateneriya, Sonu Choudhary. A Comprehensive Review on Breast Cancer. Asian J. Nur. Edu. & Research 2(1): Jan.-March 2012; Page 28-32.

7.      https://en.wikipedia.org/wiki/Letrozole.

8.      ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology. International Conference on Harmonization, IFPMA, Geneva; 2005.

9.      ICH Q2A. Text on Validation of Analytical Procedures, International Conference on Harmonization. Geneva; 1994.

10.   ICH Q2A. Text on Validation of Analytical Procedures, International Conference on Harmonization. Geneva; 1995.

11.   Sura, R. S., CVS, S., & rachamalla, S. S. (2022). Bioanalytical RP-HPLC Method Development And Validation Of Clopidogrel Bisulfate In Wistar Rat Plasma And Its Application To Pharmacokinetic Study.  International Journal of Applied Pharmaceutics, 14(1), 106–111. https://doi.org/10.22159/ijap.2022v14i1.43328

 

 

 

 

Received on 17.10.2022       Modified on 19.11.2022

Accepted on 22.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(2):103-107.

DOI: 10.52711/2231-5675.2023.00018