Application of Area Under Curve Technique for UV- Spectrophotometric Determination of Luliconazole in Bulk and Pharmaceutical Formulation


Mansi J. Chaudhari1, Suraj R. Chaudhari2, Shailesh S. Chalikwar3, Atul A. Shirkhedkar4*

1M. Pharm.  Student, Department of Quality Assurance, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule (MS) 424 405

2Assistant Professor, Department of Pharmaceutical Chemistry, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule (MS) 424 405

3Professor and Head, Department of Quality Assurance, R. C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule (MS) 425 405

4*Vice-Principal and Head, Department of Pharmaceutical Chemistry, R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur, Dhule (MS) 425 405

*Corresponding Author E-mail:



A simple, rapid, accurate and economical UV- spectrophometric method has been developed for estimation of Luliconazole in bulk and formulation using area under curve (AUC) technique. The method applied was an area under curve (AUC) in which area under curve was integrated in the wavelength range of 279 – 305 nm. The λ max of Luliconazole in methanol was found to be 295 nm. The drug follows linear in the concentration range 3-18 µg/mL with correlation coefficient value 0.997. The proposed method was applied for qualitative and quantitative estimation of Luliconazole in pharmaceutical formulation and results were found in good agreement with the label claimed. This developed method can be used for routine analysis of Luliconazole in bulk and formulation.


KEYWORDS: Luliconazole, UV-Spectrophotometry,   Area under Curve, Validation, ICH guidelines




Luliconazole (LCZ) belongs to imidazole class of drug that possesses a wide spectrum of antifungal activity and is very potent against dermatophytes. Luliconazole is chemically, [(2E)-2-[(4R)-4-(2, 4-dichlorophenyl)-1, 3-dithiolan-2-ylidene]-2-imidazol-1-ylacetonitrile] (Figure 1). The molecular formula is C14H9Cl2N3S2 with a molecular weight of 354.28 and melting point in the range 121-125˚C [1-2]. Luliconazole is a used for the treatment of interdigital tinea pedis, tinea cruris, and tinea corporis [3-4].




Figure 1: Chemical Structure of Luliconazole


The mode of action of Luliconazole is against dermatophytes is unknown, Luliconazole appears to inhibit ergosterol synthesis by inhibiting the enzyme lanosterol demethylase. Inhibition of this enzyme’s activity by azoles results in decreased amounts of ergosterol, a constituent of fungal cell membranes, and a corresponding accumulation of lanosterol [5-7]. Luliconazole may be metabolized by CYP2D6 and 3A4 [8]. The literature survey revealed that very few analytical methods were reported like simple and rapid stability-indicating liquid chromatographic method has been developed and validated for Luliconazole [9], UV Spectrophotometric method for the estimation of Luliconazole in marketed formulation [10].


To our notice, so far no UV- spectrophotometric method using Area Under Curve (AUC) has been reported for the determination of Luliconazole in bulk and formulation. Hence an attempt has been made to develop new UV spectrophotometric (AUC) method for estimation of LCZ in bulk and pharmaceutical formulations. Further, to validate developed method for accuracy, precision, repeatability, and ruggedness as per ICH guidelines [11].



Chemical and Reagent:

Luliconazole bulk powder, the formulation was purchased from the local market, Analytical grade methanol, and calibrated glass wares were employed throughout the work.



For present analysis, a double beam UV-VIS Spectrophotometer (UV-2450, Shimadzu, Japan) was used, which was linked to computer installed with spectra manager software UV Probe 2.21 with 10 mm quartz cells. The spectra were obtained with following set of instrumental parameters: wavelength range: 400 - 200 nm; scan speed: medium; sampling interval: 10 nm; band width: 1.0 nm; spectral slit width: 1 nm. An electronic balance (Model Shimadzu AUX 120) was used for weighing purpose. Micropipette (100 – 1000 µL) was also used.


Preparation of Stock Standard Solution:

The stock standard solution was prepared by accurately weighing and dissolving 10 mg LCZ in 100 mL of methanol to achieve final concentration 100 μg/mL.


Area under Curve UV-Spectrophotometric Method:

For the selection of analytical wavelength range, from the stock standard solution, an appropriate volumes 0.3 – 1.8 mL were transferred into a series of 10 mL volumetric flasks, followed by their volume make up to obtain final concentration ranging from 3 to 18 μg/mL and scanned in the spectrum mode from 200 nm to 400 nm. From the spectrum of LCZ, AUC in the wavelength range of 279- 305 nm was selected for the analysis. The calibration curve was prepared in the concentration range of 3-18 µg/ml. Concentrations of sample solution were determined using calibration curve.


Preparation of Sample Solution:

For analysis of commercial formulation 10 mg cream of Luliconazole was accurately weighed, transferred into 100 mL volumetric flask containing 50 mL of methanol, sonicated for 20 min, and the solution was diluted up to 100 mL with the same solvent and filtered through Whatman filter paper (No. 41). From the filtrate, an appropriate volume was taken and diluted with methanol to get the final concentration of 12 µg/mL for all the methods. The resulting solutions were scanned using UV-Spectrophotometer in the range of 400- 200 nm. The amounts of drug estimated using various proposed methods as determined from respective linearity equations.



Figure 2: UV-Spectrum of LCZ in methanol showing selection of wavelength for determination of LCZ

Validation of Methods:

The proposed method was validated as per ICH guidelines. The solutions of the drugs were prepared as per the earlier adopted procedure given in the experiment.



Different aliquots of Luliconazole in the range 0.3-1.8 ml was transferred into series of 10 ml volumetric flasks and the volume was made up to the mark with methanol to get concentrations 3, 6, 9, 12, 15, and 18 μg/ml, respectively. The solutions were scanned on spectrophotometer in the UV range 200-400 nm. The two wavelengths 279 and 305 nm was selected for the determination of the Area Under Curve (AUC). The calibration plot was constructed as Area Under Curve v/s concentration.



Figure 3: Linearity curve for Luliconazole



To the pre analyzed sample solutions, a known amount of the standard stock solution was added at different levels i.e. 80%, 100% and 120%. The solutions were reanalyzed by proposed method.



Method precision was performed as intra-day and inter-day deviation. It was studied using concentration 9, 12 and 15 μg/mL of LCZ; analyzed it for three times on the same day for intra-day studies while it was analyzed for the three different days over a period of week for inter-day studies.



The sensitivity of LCZ measurement was determined in terms of DL and QL denoted as limit of Detection and Quantification, respectively. The DL and QL was calculated as per 3.3*N/B and 10*N/B equations, respectively; wherein, ‘N’ denoted standard deviation of absorbance, amplitude and peak areas of LCZ (n = 3), taken as a measure of noise, and ‘B’ signifies the slope of corresponding calibration curve.



Repeatability was determined by analyzing 12µg/ml concentration of Luliconazole solution for six times.



The ruggedness of the developed methods was performed by using 12 μg/mL solution of LCZ through evaluation of aliquots from homogenous lots, performed by two different analysts under similar operational and environmental conditions for all methods.



In methanol, LCZ shows linearity in the concentration range of 3 - 18 μg/mL. The maximum absorbance (λ max) wavelength range and correlation coefficient for method given in Table 1. 


Table 1: Optical characteristics and linearity data of Luliconazole



Linearity range (μg/mL)


Selected range (nm) for AUC






Correlation coefficient


Limit of detection (μg)


Limit of quantitation (μg)




These two method, inter- and intra-day precision was studied (% RSD less than 2), and accuracy of these method was determined by calculating mean % recovery at 80, 100 and 120 % level. The results of accuracy, repeatability, and ruggedness studies are represented in Table 2.Pharmaceutical formulation of LCZ were analyzed. The amounts of LCZ in formulation were determined by these method; the results are shown in Table 3.



Table 2: Validation Parameters




80 %


100 %


120 %


Precision ( % RSD)

Intraday (n=3)

0.3 – 1.0

Interday (n=3)

0.5 – 0.7

Repeatability % RSD  (n=6)


Ruggedness (n=6) % RSD





n= number if determinations




Table 3: Analysis of Pharmaceutical formulation


% Amount found








The results of proposed study indicate that the proposed UV spectrophotometric AUC method is simple, rapid, precise and accurate. The developed UV spectrophotometric AUC method was found suitable for determination of Luliconazole in marketed formulation without any interference from the excipients. Statistical analysis proves that the method is repeatable and selective for the analysis of Luliconazole. It can therefore be conclude that the use of the method can save time and it can be used in small laboratories with accurate and wide linear range.



Authors are thankful to Principal of R. C. Patel Institute of Pharmaceutical Education and Research Shirpur, Dist: Dhule (MS) 425 405 for providing necessary laboratory facility.



Authors do not have conflict of interest for this manuscript.



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Received on 30.12.2017          Accepted on 14.01.2018        

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(1):45-48. 

DOI:   10.5958/2231-5675.2018.00008.X