Authors: Mahima Salian, Dr. Jaya Agnihotri
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Simultaneous equation technique has devised a simple, rapid, accurate, precise, and cost-effective spectrophotometric strategy for simultaneous estimation of Fluconazole and Thymol. Fluconazole and Thymol had absorbance maxima at 261 and 273 nm, respectively; hence absorbance was measured at these wavelengths for fluconazole and Thymol estimate. Fluconazole and Thymol followed Beer-Lambert\'s rule in concentration ranges of 20-140 g/ml and 20-100 g/ml, respectively. The technique was developed and verified in compliance with ICH guidelines and may be used to estimate Fluconazole and Thymol concentrations within formulations.
Spectrometry is concerned with equipment that detects or emits electromagnetic radiation as a result of its interaction with materials. The measurement of electromagnetic radiation absorbed by atoms, molecules, or ions of certain wavelengths is known as absorption spectrometry .
The quantity of absorption is determined by the wavelength of the light and the compound's structure. Radiation absorption occurs when electrons in lower energy orbitals are stimulated into higher energy orbitals. Because this is an electron transition phenomenon, UV is sometimes referred to as electronic spectroscopy [2, 3]. UV visible spectrophotometry is a technology that is commonly used in pharmaceutical analysis. It entails measuring the quantity of ultraviolet (190-380nm) or visible (380800nm) radiation absorbed by a material in solution using equipment that measures the ratio or a function of the ratio of the intensity of two light beams in the UVvisible area .
Fluconazole is an antifungal medicine that is a synthetic triazole derivative with the chemical formula 2-(2, 4-difluorophenyl)-1, 3-bis (1H-1, 2, 4-triazol-1-yl).-2-propanol (Fig 1). It is effective against a wide range of systemic and superficial fungal diseases . Fluconazole is a BCS Class II medication with strong permeability and low solubility. Hence it is insoluble in water and highly soluble in organic solvents. Fluconazole interacts with the 14-demethylase cytochrome P-450 enzyme, which catalyses the conversion of lanosterol to ergosterol. It inhibits the synthesis of ergosterol, which is required for fungal cell membrane development, and increases cellular permeability . Thymol, also known as 2-isopropyl-5-methylphenol, (Fig 2) is a monoterpene phenol that is prevalent in plants such as Thymus vulgaris. Due to the deprotonation of phenol, it is highly soluble in alcohols, alkaline solutions, and other organic solvents, but it is only slightly soluble in water at neutral pH and absorbs the most UV radiation at 273 nm.
Thymol disrupts cell wall or membrane integrity and interferes with ergosterol biosynthesis . To treat fungal infections caused by Candida albicans, a combination of fluconazole and thymol has been developed. Since synthetic drugs are resistant to Candida albicans, combining them with natural constituents produces synergistic efficacy. The literature search revealed that, although a promising combination, there is no method available for simultaneous estimation of thymol and fluconazole from dosage form. Therefore, developing a UV spectrophotometric method for the simultaneous estimation of thymol and fluconazole is challenging. Therefore, the aim of the present work was to develop & validate a UV spectroscopic method for simultaneous estimation of Fluconazole and thymol from Spray formulation.
II. Materials and Methods
A. Chemical and instrument
The standard drug Fluconazole was received as gift sample from FDC (Mumbai) and Thymol from Powder Pack Chem India. Ethanol was purchased from Vishal chem. Kolidone VA64 polymer was obtained from Ashland India Pvt Ltd. Ethyl Cellulose was obtained from Vishal chemicals. All other excipients were procured from vishal chemicals. All solvents and reagent used were of analytical grade. Over the range of 200-400 nm, a UV-spectrophotometer UV-1800 (Shimadzu, Japan) with a spectral bandwidth of 2 nm and 10 mm matched quartz cells was utilised to create an analytical procedure.
2. Selection of wavelength for analysis of Fluconazole and Thymol:
Fluconazole (100 mg) and Thymol (100 mg) were accurately weighed and transferred to a 100 mL volumetric flask, dissolved, and diluted to the mark with ethanol to generate a standard solution with Fluconazole and Thymol concentrations of 1000 ppm each. The concentration of the drugs 100µg/ml each in ethanol was generated from this stock solution and scanned using a spectrophotometer within the wavelength range of 200 to 800nm against ethanol as a blank(fig 3).
3. Calibration of Curve of Fluconazole and Thymol
A series of calibrated 10mL volumetric flasks were taken and appropriate aliquots of stock solution of Thymol and fluconazole were withdrawn and diluted up to 10mL with water, Different dilutions were made from this stock solution, ranging from 20µg/ml to 140µg/ml for Fluconazole. The absorbance was measured at absorption maxima 261 nm for Fluconazole against the reagent blank prepared in a similar manner without Fluconazole with Ethanol (Fig4). The same procedure was applied for Thymol stock solution solutions having concentrations of 20μg/mL to 100μg/mL and absorbance was measured at 273nm, against a reagent blank prepared in a similar manner without Thymol with Ethanol (fig 5).
4. Simultaneous Equation Method [8-10]:
If a sample contains two absorbing drugs, each of which absorbs at the λmax of the other, it may be possible to determine both drugs simultaneously using multicomponent analysis UV Spectrophotometric ‘Simultaneous Equation Method. Two simultaneous equations (in two variables Cx and Cy) were formed using these Absorptivity coefficient values. Concentrations in the sample were obtained by using the following equations.
An analytical procedure's ability to be linear is its ability to rationally demonstrate that the observed absorbance is proportionate to the concentration of a sample containing the analyte. Fluconazole's linearity was investigated at 261 nm using seven different concentrations that ranged from 20 - 140 µg/mL. Thymol's linearity was also investigated using five different concentrations that ranged from 20 - 100 µg/mL. Plotting concentration vs absorbance allowed for the creation of the calibration curves. Fitting to the equation y = mx + c yielded the regression's slope, intercept, and correlation coefficient values (Table 1).
Precision is defined as the closeness of the readings obtained by multiple measurements of the same sample under prescribed conditions (Table 3 & Table 4). Intraday and interday precision are considered for the precision studies. Intraday Precision: The absorbance of fluconazole and thymol solution samples at concentrations of 20, 60, and 100 g/mL was acquired three times on the same day and the % RSD was determined. Interday Precision: The absorbance of Fluconazole and Thymol sample solutions at concentrations of 20, 60, and 100 g/mL was tested on three different days and the percentage RSD was calculated.
The accuracy of an analytical approach displays the degree of agreement between the values considered as conventional true values and the value discovered. The method's accuracy was established by conducting recovery experiments. The recovery investigations were carried out by mixing the previously analysed formulation sample solution with the reference medication solution. The data had to be accurate within 2% standard deviation (SD) of nominal values and precise within 2% relative standard deviation (RSD) to be accepted (Table 5).
4) Limit of Detection (L.O.D)
The lowest concentration of analyte in a sample that can be detected but not always quantitated as an accurate number is referred to as the detection limit of an analytical procedure. The standard curve was used to determine the limit of detection.
The authors are thankful to FDC India and Powder pack chem India, for providing drug samples and Principal of H.K College of Pharmacy, for providing facilities to carry out this research work.
V. CONFLICT OF INTEREST
The authors report no conflicts of interest
The proposed method, which uses a spectrophotometric method of analysis that is less expensive and simpler than methods like chromatography and electrophoresis, can be used for routine quality control analysis of Fluconazole and Thymol pharmaceutical dosage forms. The developed method was found to be sensitive, accurate, precise, reproducible, and linear over the concentration range studied.
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