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ORIGINAL ARTICLE
Year : 2010  |  Volume : 1  |  Issue : 3  |  Page : 348-353 Table of Contents     

Spectrophotometric methods for the determination of letrozole in bulk and pharmaceutical dosage forms


Department of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha - 760 010, India

Date of Web Publication10-Nov-2010

Correspondence Address:
Sasmita Kumari Acharjya
Department of Pharmaceutical Analysis and Quality Assurance, Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha - 760 010
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0110-5558.72425

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   Abstract 

Ultraviolet (UV), first derivative, second derivative, and AUC-spectrophotometric methods for the determination of letrozole in pharmaceutical formulations have been developed. For UV-spectrophotometry, the standard solutions were measured at 240.0 nm. The linearity ranges were found to be 0.25-20.0 μgml−1 in methanol and the regression equation was A=1.20×10−1 C+2.22×10−2 (r 2 =0.9994). For the first derivative spectrophotometry, the response (dA/dλ) of standard solutions was measured at 224.0 nm. The calibration curve was constructed by plotting dA/dλ values against concentrations 0.25-20.0 μgml−1 , of letrozole. The regression equation of the linear calibration graph was calculated as D 1 =3.89×10−3 C+1.85×10−4 (r 2 =0.9987). For the second derivative spectrophotometry, the response (d 2 A/dλ2 ) of standard solutions was measured at 241.0 nm. The calibration curve was constructed by plotting d 2 A/dλ2 values against concentrations 0.5-20.0 μgml−1 of letrozole standards in methanol. The regression equation of the linear calibration graph was calculated as D 2 =-1.59×10−3 C -4.66×10−4 (r 2 =0.9985). The AUC-spectrophotometric method was based on the calculation of Area under Curve (AUC), for analysis of letrozole in the wavelength range of 235.0-245.0 nm. The calibration curve was constructed by plotting AUC values against concentrations 0.25-20.0 μgml−1 , of letrozole. The regression equation of the linear calibration graph was calculated as AUC=1.132C+0.2153 (r 2 =0.9994). The methods were validated by following the analytical performance parameters suggested by the International Conference on Harmonization (ICH). All validation parameters were within the acceptable range. The developed methods were successfully applied to estimate the amount of letrozole in pharmaceutical formulations.

Keywords: AUC-spectrophotometry, derivative-spectrophotometry, letrozole, UV-spectrophotometry


How to cite this article:
Acharjya SK, Mallick P, Panda P, Kumar K R, Annapurna M M. Spectrophotometric methods for the determination of letrozole in bulk and pharmaceutical dosage forms. J Adv Pharm Technol Res 2010;1:348-53

How to cite this URL:
Acharjya SK, Mallick P, Panda P, Kumar K R, Annapurna M M. Spectrophotometric methods for the determination of letrozole in bulk and pharmaceutical dosage forms. J Adv Pharm Technol Res [serial online] 2010 [cited 2020 Dec 3];1:348-53. Available from: https://www.japtr.org/text.asp?2010/1/3/348/72425


   Introduction Top


Letrozole, 4-[(4-cyanophenyl)-(1, 2, 4-triazol-1-yl)methyl]benzonitrile, is a selective nonsteroidal inhibitor of the aromatase (oesterogen synthetase) system, which is used for the treatment of esterogen-dependent breast cancers. [1] Letrozole is readily and completely absorbed from the gastrointestinal tract. It is slowly metabolized in the liver to an inactive carbinol metabolite, which is then excreted as glucoronide in the urine. [2]

On a detailed literature survey, it was found that letrozole could be estimated by spectrophotometry, [3],[4] High-performance liquid chromatography (HPLC), [5],[6],[7],[8],[9],[10] the microarray approach, [11] capillary gas chromatographic method with flame ionization detector, [12] and by gas chromatography - mass sprectrometry (GC / MS) [13] methods.

Experimental

Chemicals and reagents


The Letrozole working standard was kindly provided by Alembic Ltd., (Vadodara, India) and was used as received. A commercial tablet formulation was purchased from the local market. Analytical reagent grade methanol was used for the preparation of solutions.

Instrumentation

A double beam UV-VIS spectrophotometer (UV-1800, Shimadzu, Japan) connected to a computer loaded with a 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 - 400 nm; scan speed: medium; sampling interval: 0.2 nm; derivative mode: 1 D (first order derivative, dA / dλ) and 2 D (second order derivative, d 2A / dλ2); band width (Δλ): for 1 D and 2 D, 10.0 nm; spectral slit width: 1 nm. All weights were taken on an electronic balance (Denver, Germany).

Preparation of standard stock solution

The standard solution of letrozole was prepared by dissolving 10 mg of the drug (accurately weighed) in methanol and diluted to 100 ml with methanol to obtain a final concentration of 100 μg ml -1 . This stock solution was used to prepare further dilutions of standard solutions.

Method I

UV-spectrophotometry


Series dilutions of the stock solution were made by pipetting out 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 ml stock solution into separate 10 ml volumetric flasks and diluting to volume with methanol to produce concentrations ranging from 0.25 - 20.0 μgml -1 . The above-mentioned solutions were scanned over the range of 400 nm to 200 nm against the blank. The λmax was found to be at 240.0 nm. The calibration curve was constructed by plotting the concentration (0.25 - 20.0 μgml -1 ) versus absorbance, at 240.0 nm.

Method II

First-derivative spectrophotometry


The spectra obtained in method I were derivatized to get first-order derivative spectra and the response (dA / dλ) of the spectra were measured at 224.0 nm, and then the calibration curve was constructed by plotting the concentration (0.25 - 20.0 μgml -1) versus response (dA / dλ), at 224.0 nm.

Method III

Second-derivative spectrometry


The spectra obtained in method I were derivatized to get second-order derivative spectra and the response (d 2A / dλ2 ) of the spectra were measured at 241.0 nm, and then the calibration curve was constructed by plotting the concentration (0.5 - 20.0 μgml−1 ) versus response (d 2A / dλ2 ), at 241.0 nm.

Method IV

Area under curve


The AUC (area under curve) method is applicable where there is no sharp peak or when broad spectra are obtained. It involves calculation of the integrated value of absorbance with respect to the wavelength between the two selected wavelengths λ1 and λ2 . The 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. This wavelength range is selected on the basis of repeated observations, so as to get the linearity between the area under curve and concentration. The spectra obtained in method I were used to calculate the AUC. The calibration curve was constructed by plotting the concentration (0.25 - 20.0 μg ml -1 ) versus AUC.

Estimation of Letrozole in Tablets

For the analysis of the pharmaceutical dosage form, a total of 20 tablets were weighed and finely powdered. A portion of the powder, equivalent to about 10 mg letrozole was weighed accurately and transferred into a 100 ml volumetric flask and 50 ml methanol was added. After ultrasonic vibration for 30 minutes, the mixture was diluted to volume with methanol and filtered through Whatman filter paper (No. 41). Appropriate dilution was made into 5.0 μgml -1 with methanol from the stock solution for all the methods, and the amounts of letrozole were determined. The percent labeled claim and Standard Deviation (S.D) were calculated.

Validation of Methods

Linearity


For all the methods, six-point calibration curves were prepared on three different days. The results obtained were used to calculate the equation of the line by using linear regression by the least-squares regression method.

Precision

The intra-day and inter-day precisions of the proposed spectrophotometric methods were determined by estimating the corresponding response thrice on the same day and on three different days, over a period of one week, for three different concentrations of letrozole (2.5, 5.0, and 10.0 μgml -1 ) and the results were reported in terms of relative standard deviation.

Accuracy


This parameter was evaluated by the percent recovery studies at concentration levels of 80, 100, and 120%, which consisted of adding a known amount of letrozole reference material to a prequantified sample solution. An aliquot of the sample solution containing letrozole at 5.0 μgml -1 was transferred to three 10 ml volumetric flasks containing, 4.0, 5.0, and 6.0 μgml -1 letrozole reference solutions, respectively. The contents were mixed and diluted to volume, in order to obtain the final concentrations of 9.0, 10.0, and 11.0 μgml -1 letrozole. The recoveries were verified by the estimation of drugs in triplicate preparations, at each specified concentration level. The spectra were recorded in the UV range and then analyzed. The results were reported in terms of % recovery.

Specificity


The results of the tablet solution showed that there was no interference of excipients when compared with the working standard solution. Thus, the methods were said to be specific.

Robustness

The robustness of the proposed methods was tested by changing parameters such as wavelength range and slit width. None of these variables significantly affected the absorbance of the drugs, indicating that the proposed methods could be considered as robust.

Ruggedness


Ruggedness of the proposed methods was determined by analyzing aliquots from a homogenous slot (5.0 μgml -1 ) in different laboratories, by different analysts, using similar operational and environmental conditions. The results are reported in terms of % RSD.


   Results and Discussion Top


[Figure 1],[Figure 2], and [Figure 3] show overlaid UV-spectrophotometric (0.25 - 20.0 μgml -1 ), first-derivative (0.25 - 20.0 μgml -1 ), and second-derivative (0.5 - 20.0 μgml -1 ) absorption spectra of letrozole, respectively, and the spectra are found to be similar in nature and overlapping. [Figure 4] shows the absorption spectrum of letrozole (5.0 μgml -1 ) in methanol for method IV. The optical characteristics of letrozole have been calculated by the proposed methods and are presented in [Table 1].
Figure 1: Absorption spectrum of letrozole in methanol (0.25 - 20.0 μgml-1)

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Figure 2: First-derivative absorption spectrum of letrozole in methanol (0.25 - 20.0 μgml-1): (a) Normal view (b) Large view

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Figure 3: Second-derivative absorption spectrum of letrozole in methanol (0.25 - 20.0 μgml-1): (a) Normal view (b) Large view

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Figure 4: Absorption spectrum of letrozole (5.0 μgml-1) in methanol [235.0 - 245.0 nm range was selected for Method-IV]

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Table 1: Optical characteristics of letrozole

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Stock solution of the drug was prepared in methanol and further dilutions were carried out with methanol, 0.1N HCl, 0.1N NaOH, and with water separately, to produce concentrations ranging from 0.25 - 20.0 μgml -1 . However, in the present study, dilutions carried out with methanol were selected because the drug solutions were stable and the Beer-Lambert's law was followed properly [Table 2]. From the calibration curve [Figure 5], it was observed that with an increase in letrozole concentration, the responses increased. In Method I, the λmax was found to be at 240.0 [Figure 1]. Hence, the study was carried out at 240.0 nm, where the Beer- Lambert's law was followed properly.
Figure 5: Calibration curves of letrozole in methanol (Method I, II, III, and IV)

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Table 2: Comparison of absorbance of letrozole in different solvents

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Derivative spectrophotometry is an analytical technique for the enhancement of sensitivity and specificity in qualitative and quantitative analysis of various compounds, including pharmaceuticals. Hence, methods II and III have been carried out for letrozole. For Method II [Figure 2], 224.0 nm is selected because at 208.0 nm the peaks are distorted and the maximum wavelength of the peaks as well as the zero crossing point do not remain constant, and at 247.0 nm, the Beer-Lambert's law is not followed properly. For Method III [Figure 3], the wavelength 241.0 nm is selected, because the zero crossing point and maximum wavelength do not remain constant for each concentration at 213.0 nm, and at 251.0 nm, the Beer- Lambert's law is not followed properly. In Method IV [Figure 4], the study has been carried out at two wavelength ranges, that is, 235.0 - 245.0 nm and 230.0 - 250.0 nm, but good linearity range was obtained at the wavelength range of e 235.0 - 245.0 nm.

Tablets were analyzed and the amount of the drug determined by the proposed methods; it was in good agreement with the label claim [Table 3]. It was also observed that there was no significant difference in the content of letrozole obtained by using the different proposed spectrophotometric methods.
Table 3: Assay results of letrozole in pharmaceutical dosage form (Tablet-2.5 mg) by using the proposed spectrophotometric methods

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The recoveries of letrozole, which was evaluated by the percent recovery studies at concentration levels of 80, 100, and 120% were found to be in the acceptable range [Table 4]. Excipients used in the formulation did not interfere with the response of the drug at its analytical wavelengths. Also, no significant change in response to letrozole was observed by changing parameters, such as, wavelength range and slit width. The intra-day and inter-day precision values (%RSD) were calculated [Table 5] and were lying in the acceptable range for letrozole. Ruggedness of the proposed methods were determined with the help of two different analysts and the results were evaluated by calculating the %RSD value, and were found to be within the range [Table 6]. Hence, the proposed methods were precise, specific, accurate, rugged, and robust for the estimation of letrozole in bulk and pharmaceutical formulations.
Table 4: Results for accuracy studies of letrozole by proposed spectrophotometric methods

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Table 5: Results for precision studies of letrozole by proposed spectrophotometric methods

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Table 6: Ruggedness data of letrozole (5.0 μg ml-1) by proposed methods

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   Conclusions Top


The four methods that were developed for the determination of letrozole were based on different analytical techniques, zero-derivative, first-derivative, and second-derivative spectrophotometry, and the AUC method. All the methods were validated and found to be simple, sensitive, accurate, and precise. Hence, all the methods could be used successfully for the routine analysis of the pharmaceutical dosage forms of letrozole.


   Acknowledgments Top


The authors are grateful to the authorities of M/s. Roland Institute of Pharmaceutical Sciences, Department of Pharmaceutical Analysis and Quality Assurance, for providing the necessary facilities to carry out the research work and to Alembic Ltd., (Vadodara, India) for providing the gift sample of the pure drug.

 
   References Top

1.Lamb HM, Adkins JC. Letrozole: A review of its use in postmenopausal women with advanced breast cancer. Drugs 1998;56:1125-40.  Back to cited text no. 1
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2.Iveson TJ, Smith IE, Ahern J, Smithers DA, Trunet PF, Dowsett M. Phase I study of the oral nonsteroidal aromatase inhibitor CGS 20267 in healthy postmenopausal women. J Clin Endocrinol Metab 1993;77:324-31.  Back to cited text no. 2
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3.Mondal N, Pal TK, Ghosal SK. Development and validation of a spectrophotometric method for estimation of letrozole in bulk and pharmaceutical formulation. Pharmazie 2007;62:597-8.  Back to cited text no. 3
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4.Ganesh M, Kamalakannan K, Patil R, Upadhyay S, Srivatsava A, Sivakumar T, et al. A validated UV spectrophotometric method for the determination of letrozole in bulk and solid dosage form. Rasyan J Chem 2008;1:55-8.  Back to cited text no. 4
    
5.Mondal N, Pal TK, Ghosal SK. 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 Pol Pharm 2009;66:11-7.  Back to cited text no. 5
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6.Marfil F, Pineau V, Sioufi A, Godbillon SJ. High-performance liquid chromatography of the aromatase inhibitor, letrozole, and its metabolite in biological fluids with automated liquid-solid extraction and fluorescence detection. J Chromatogr B Biomed Appl 1996;683:251-8.  Back to cited text no. 6
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7.Laha TK, Patnaik RK, Sen S. Reverse phase high performance liquid chromatographic method for the analysis of letrozole in pharmaceutical dosage forms. Indian J Pharm Sci 2008;70:401-3.  Back to cited text no. 7
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8.Zarghi A, Foroutan SM, Shafaati A, Khoddam A. HPLC determination of letrozole in plasma using fluorescence detection: Application to pharmacokinetic studies. Chromatographia 2007;66:747-50.  Back to cited text no. 8
    
9.Sekar V, Jayaseelan S, Subash N, Udhaya KE, Perumal P, Venkatesh RP. Bioanalytical method development and validation of letrozole by RP-HPLC method. Int J Pharm. Res Dev 2009;1:1-7.  Back to cited text no. 9
    
10.Pfister CU, Duval M, Godbillon J, Gosset G, Gygax D, Marfil F, et al. Development, application and comparison of an enzyme immunoassay and a high-performance liquid chromatography method for the determination of the aromatase inhibitor CGS 20,267 in biological fluids. J Pharm Sci 1994;83:520-4.  Back to cited text no. 10
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12.Berzas JJ, Rodriguez J, Contento AM, Cabello MP. Determination of drugs used in advanced breast cancer by capillary gas chromatography of pharmaceutical formulations. J Separation Sci 2003;26:908-14.  Back to cited text no. 12
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]


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