INTRODUCTION:

Fenticonazole Nitrate (FTZ) is an Imidazole antifungal drug used in the treatment of vulvovaginal candidiasis. It is active against the range of organism including dermatophyte pathogens, Malassezia furfur, and Candida albicans¹. The interesting mechanism of action of Fenticonazole is inhibition of the secretion of protease acid by Candida albicans, damage to the cytoplasmic membrane; and by blocking cytochrome oxidases and peroxidises². Fenticonazole Nitrate chemically is 1-[2(2,4-dichlorophenyl)-2 {[4 (phenyl sulfanyl) phenyl]methoxy}ethyl]1H-imidazole³. The chemical structure of Fenticonazole Nitrate is shown in Figure 1. Fenticonazole is official reported in EP with their characteristics, Identification test, Assay and Impurity detection⁴. Some literature of Fenticonazole is also exist in Merck index and Martindale^5,6.The information of related substance with their identification test are reported in BP⁷. In literature, few liquid chromatography procedures have been reported for the analysis of Fenticonazole Nitrate^8-12. The present UV-Spectrophotometric methods were designed for estimation of Fenticonazole Nitrate using Hydrotropy approach.

Figure 1: The Chemical Structure of Fenticonazole Nitrate

Hydrotropy is defined as increasing the solubility of different substances in water, and it could be possible by adding large quantities of second solvent (hydrotropic agents)¹³. Specific hydrotropic solutions have been used to solubilize the poorly water soluble products. Sodium benzoate, niacinamide, sodium lauryl sulphate, sodium salicylate, sodium acetate, sodium citrate, and urea were used to improve the aqueous solubility of many poorly water-soluble drugs^14-16. The number of drugs was analyzed using hydrotropy approach including Dextromethorphan, Atorvastatin, Amlodipine Besylate, Gliclazide^17-20. The Area Under Curve (AUC) technique applies where no sharp spectra is obtained or broad spectrum is obtained. This requires measuring the combined AUC value between the two chosen wavelengths λ1 and λ2^21-26. The objective of this investigation is to develop an easy, rapid, precise, reproducible, eco-friendly and economical method for determination of fenticonazole nitrate using UV-spectrophotometry by analyzing absorbance and Area Under Curve (AUC) techniques.

Chemical and Reagents:

Fenticonazole Nitrate was gifted by Glenmark Pharmaceuticals ltd. Mumbai. Pluronic F127 was purchased from Sigma Aldrich Pvt. Ltd. Bangalore and Vaginal Capsule (Fenza 600mg) was purchased from local market.

Instrumentation:

· Spectrophotometer: UV-2450 Shimadzu, Japan

· Software: UV Probe 2.21

· Sample cell: 1cm quartz cuvette

· Lamp: Deuterium Lamp Wavelength range 200 – 400nm

· Spectral Slit width: 1.0nm

· Weighing Balance: Shimadzu AUX-120

EXPERIMENTAL:

Preliminary solubility studies and selection of solvent:

Solubility of FTZ was assessed in aqueous solution of different hydrotropic agent including Polyethylene Glycol, Sodium Acetate, Urea, Pluronic (F68, F127), Benzalkonium chloride, Sodium Citrate, Potassium Acetate, and Sodium Benzoate. FTZ was found be soluble in Pluronic F127 solution. The drug was found to be completely soluble in Pluronic F127 solution of various strengths such as 30%, 20%, and 10% (w/v). Minimum concentration (10% w/v) of Pluronic F127 was chosen for solubilization of drug for entire study.

Preparation of Pluronic F127 (10 % w/v) solution:

100g of Pluronic F127 was transferred in 1000ml volumetric flask containing 700ml water, sonicated for 20 minutes and volume was made upto the mark with water.

Preparation of stock standard solution:

Stock standard solution of FTZ was prepared by dissolving accurately weighed 10mg of FTZ into 50ml of Pluronic F127 (10% w/v) solution, sonicated for 15 min and further diluted upto 100ml same solvent to obtain concentration of 100µg/mL. The working standards were prepared by dilution of the stock standard solution.

Determination of λ max and calibration curve:

A fixed volume of 1.0mL of FTZ from stock solution was transferred to 10mL volumetric flask, diluted to mark with water to obtain concentration of 10µg/mL. The resultant solution was scanned in UV range (400-200nm) in 1.0cm cell against solvent blank. The spectrum showed an absorption maximum at 253nm.

In Method I absorbance at 253nm was considered for analysis while for Method II two wavelengths 242.2-261.6nm were selected for determination of Area under Curve [AUC]. Optical Characteristics of FTZ presented in Table 1. Zero order UV-spectrum showing maximum absorbance and AUC are shown in Figure 2.

Table 1: Optical Characteristics of Fenticonazole Nitrate

Parameter	Method I	Method II
Wavelength	253 nm	242.2-261.6nm
Linearity range (µg/ml)	08 - 40	08 – 40
Correlation coefficient	0.996	0.999

(Method I)

Figure 2: Method I. UV-spectrum of Fenticonazole Nitrate in Pluronic F127 (10%w/v) showing λmax

An appropriate volume of stock solution in the range of 0.8–4.0mL were transferred in series of 10mL volumetric flask, volume was made up to 10mL with Pluronic F127 (10%w/v) solution to get concentration of 8–40µg/mL and absorbance was measured at 253nm (method I) and Zero order- AUC was recorded in between the wavelength range of 242.2-261.6nm (Method II) against the blank. Calibration curves were prepared by plotting concentration versus absorbance and AUC as shown in Figure 3.

Method II)

Figure 3: Method II. UV-spectrum of Fenticonazole Nitrate in Pluronic F127 (10%w/v) showing selection of wavelength for integration of AUC

Analysis of marketed formulation:

Ten tablets were accurately weighed and average weight determined, an amount of power drug equivalent to 10 mg of Fenticonazole Nitrate was transfer into 100 mL volumetric flask containing 50ml of Pluronic F127 (10%w/v) sonicated for 15 min. The volume was made up to the mark with same solvent and filtered through 0.45µm Whatman filter paper. A suitable volume of solution was further diluted with 10% w/v Pluronic F127 solution to obtain concentration 24µg/mL of Fenticonazole Nitrate for tablet assay. These sample solutions were scanned at selected wavelengths in Method I and Method-II and the results were obtained. From the respected linear regression equations, the concentrations were determined. The procedure was repeated for six times and the results are shown in Table 2.

Table 2: Analysis of vaginal capsules

	Label Claim (mg)	Conc.(µg/ml)	% Amount Found [n=6]	± SD	% RSD
Method I	600	24	99.36	0.969	0.975
Method II	600	24	99.17	0.504	0.508

Validation:

The method was validated for accuracy, precision and ruggedness according to ICH guidelines²⁷.

Accuracy:

To estimate the accuracy of both the proposed methods, recovery studies were executed out at 80, 100 and 120% of the test concentration as per ICH guidelines. To the pre-analyzed sample solution (16µg/ml), a known amount of drug standard was added at 80, 100 and 120% and solutions were reanalyzed by proposed methods. The experiments were performed for three times at each level for each method. The results of the recovery studies are reported in Table 3.

Table 3: Validation Parameters

Precision (%RSD)
Inter-day[n=3]	0.95 - 1.13	0.78 – 1.73
Intra-day[n=3]	0.20 – 1.03	0.34 – 1.38
Repeatability [n=6]	1.02	1.22
Ruggedness (%RSD)
Analyst I [n=6]	0.97	1.43
Analyst II [n=6]	1.56	1.78
Accuracy studies
% Recovery	99.09 - 99.59	99.40 – 100.03
%RSD [n=3]	0.95 – 1.65	1.43 – 1.40
Sensitivity
DL (µg/ml)	1.63	4.89
QL (µg/ml)	1.77	5.16

Precision:

Precision of the methods were studied as intra-day, inter-day variations and repeatability. Precision was determined by analyzing the concentration of 16, 24, and 32µg/ml. To determine the degree of repeatability of the methods, statistical evaluation was carried out. Repeatability was determined by analyzing Fenticonazole concentration of 24µg/mL for six times and results reported in Table 3.

Ruggedness:

Ruggedness of the proposed method was determined by analysis of aliquots from homogenous slot by two analyst using same operational and environmental conditions and the results are reported in Table 3.

Sensitivity:

The sensitivity of proposed methods was estimated in terms of estimating Detection Limit (DL) and Quantitation Limit (QL) which were calculated using formulae “DL = 3.3 ×N/B,” and “QL = 10 × N/B” where “N” is average standard deviation of the absorbance or peak areas of the Fenticonazole (n = 3), taken as a measure of noise, and “B” is the slope of the corresponding calibration curve.

RESULTS AND DISCUSSION:

In the present investigation, hydrotropic solubilization is employed to enhance the aqueous solubility of poorly water-soluble drugs Fenticonazole Nitrate in bulk and in vaginal capsules. By selecting proper hydrotropic agents, the use of organic solvents in analysis may be decreased to a greater extent. For the solubility studies, different hydrotropic agent was tried but optimum solubility was achieved in Pluronic F127 (10% w/v) solution. In Method I and II, linearity of Fenticonazole Nitrate was found to be in the range of 08 - 40µg/mL, with correlation coefficient (r2 > 0.99). Marketed brand of Vaginal capsules were analyzed. The amounts of Fenticonazole Nitrate determined by ‘Method I’ and Method II was found to be 99.36% and 99.17, respectively. In both these methods, precision was studied as repeatability, inter and intra-day variations at three different concentrations of Fenticonazole Nitrate and % RSD was found to be less than 2. The accuracy of method was determined by calculating mean percentage recovery. It was determined at 80, 100 and 120% level and % recovery was found to be in the range 99.09% - 99.59% and 99.40% – 100.03% for method I and II respectively. The ruggedness of the methods was studied by two different analysts using the same operational and environmental conditions and % RSD found to be less than 2. DL and QL were found to be 1.63µg/ml and 4.89 µg/ml, respectively for Method I and DL and QL were found to be 1.77µg/ml and 5.16µg/ml respectively for Method II indicating adequate sensitivity of the methods.

CONCLUSION:

The proposed methods for analysis of Fenticonazole Nitrate in pharmaceutical formulations are ecofriendly; simple, precise and rapid so can be employed for routine analysis. From the proposed methods, we conclude that there is a good scope for other poorly water-soluble drugs to get solubilized by using suitable hydrotropic agents. The developed methods have advantages as organic solvents are avoided.

Method I

Method II

Figure 4: Calibration curve of Fenticonazole Nitrate

ACKNOWLEDGMENTS:

Authors are thankful to Dr. S.J. Surana, Principal, R.C. Patel Institute of Pharmaceutical Education for providing necessary facilities for this work.

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