Introduction
Luliconazole belongs to the imidazole class of medicines renowned for their potent antifungal effects, especially against dermatophytes.1 It has a wide range of antifungal activities. Its chemical formula is C14H9Cl2N3S2, and it weighs 354.28 in molecular mass. Chemically, it is known as (2E)-2-[(4R)-4-(2,4-dichlorophenyl)-1,3-dithiolan-2-ylidene]-2-imidazol-1-yl-acetonitrile.2 In its molecular structure, the E-configured double bond is adjacent to the dithiolane group. Luliconazole is a crystalline, off-white to pale yellow powder and is soluble in solvents like methanol, acetonitrile, dimethylformamide, and dimethyl sulfoxide.3 While, the precise mechanism through which luliconazole acts against dermatophytes remains uncertain, it is believed to impede the production of ergosterol by inhibiting the enzyme lanosterol demethylase Azoles, including luliconazole, inhibit this enzyme's activity, leading to reduced ergosterol levels, a vital constituent of fungal cell membranes. This disruption results in an accumulation of lanosterol.
Beclomethasone dipropionate, a potent anti-inflammatory medication, is a synthetic corticosteroid. 4 It goes by the chemical names 1,4-diene-17,21-diyldipropionate and 9a-chloro-11b-hydroxy-16b-methyl-3,20-dioxopregnane. Its molecular weight is 521.042, with a chemical formula of C28H37ClO7. This substance appears nearly white crystalline powder, with a pKa of 13.6 and a melting range of 117–120°C.5, 6 In terms of its mechanism of action, Beclomethasone dipropionate interacts with DNA at the cellular nucleus level, influencing gene transcription by either stimulating or inhibiting it. This drug functions as a factor that regulates gene expression, which means that it may change how certain genes are expressed in response to particular hormone signals.7, 8
Luliconazole and beclomethasone dipropionate is a well-known marketed as Luliford-B cream.
Materials and Methods
Chemicals and reagents
The Luliconazole working standard, with a stated potency of 98.27% on a dried basis, was obtained from the Central Drug Testing Laboratory (CDTL) in Mumbai. Similarly, the Beclomethasone dipropionate working standard, with a claimed potency of 98.13%, was also sourced from the Central Drug Testing Laboratory in Mumbai. For the Luliford-B cream, a marketed formulation by Leeford Healthcare Ltd., the composition includes Luliconazole at a concentration of 1% w/w and Beclomethasone dipropionate at a concentration of 0.025% w/w. Acetonitrile (ACN) and Methanol of high-performance liquid chromatography (HPLC) grade was procured from Merck Life Science. Glacial Acetic Acid was sourced from Finar Chemicals located in Gujarat. Ultra-purified water of HPLC grade was obtained from Milli-Q®. Additionally, a high-flow nylon membrane filter with a pore size of 0.45µm from Axiva Sichem Pvt. Ltd.
Instrumentation
UV-visible spectrometer from Lab India. All UV spectrophotometric measurements were performed using Win Lab software. Chromatography was performed using a Thermo Scientific Dionex Ultimate 3000 with Chromeleon 7.2.6 software and LC instrument control. The Sartorius Analytical Balance was used for all weighing. The column utilized had a particle size of 4.6mm, 25cm and 5µm and was inert enough to last.
Choice of diluent (solvent)
Based on the solubility and chemical nature of luliconazole and beclomethasone dipropionate, the HPLC grade methanol was selected as a diluent for preparing standard and sample solutions.
Preparation of Standard Solution
Transferred approximately 10mg of Luliconazole working standard in a 10ml volumetric flask, sonicated it to dissolve it with an adequate amount of diluent, and then adjusted the volume (1000ppm standard stock solution). Weighed out around 10 mg of Beclomethasone dipropionate working standard, dissolved it with enough diluent by sonication, and adjusted the volume in a 100ml flask (100ppm standard stock solution). Then take 2 ml of 1000 ppm of stock solution of luliconazole and 5ml of 100 ppm of stock solution of beclomethasone dipropionate and make up to 20 ml with diluent (100ppm of luliconazole and 2.5ppm of beclomethasone dipropionate).
Analysis of marketed formulation
Transferred equivalent 1g of cream (equivalent 1%w/w luliconazole and 0.025%w/w beclomethasone dipropionate) to 100 ml flask, sonicated for 15 minutes with an adequate amount of diluent, and then filtering through a 0.45µ nylon filter to create volume.
Method validation
The method that was created underwent validation in compliance with the standards established by aspects including system suitability, linearity, precision (both repeatability and intermediate precision), accuracy, assay, robustness, limit of detection (LOD), limit of quantification (LOQ), and a stability indicating study. These evaluations were carried out following specific procedures. 8, 9
System suitability
The table labelled as Table 1 presents the parameters for the system suitability test regarding luliconazole and beclomethasone dipropionate in the developed method.10 The percent relative standard deviation (% RSD) for retention time, tailing factor, theoretical plate count, peak area, and for both luliconazole and beclomethasone dipropionate was determined to be within the specified limit of 2%. This outcome suggests that the system is appropriate and functions suitably.11
Additionally, the number of theoretical plates and tailing factor for both compounds met the acceptance criteria of being ≥ 2000 and ≤ 2.0, respectively. This demonstrates efficient column performance and an optimal composition of the mobile phase.12
Table 1
Result from system suitability study
Linearity
The linearity of the analytical method was assessed using linear regression analysis, employing the least squares method.13 This evaluation involved the preparation of standard solutions for luliconazole and beclomethasone dipropionate at various concentration levels. After measuring the peak area of the final solutions, the peak area was plotted against the amount present of each drug to create a calibration curve. (Figure 7, Figure 8). In the range, the reaction was discovered to be linear 5-150µg/ml for luliconazole and 0.4-4.8µg/ml for beclomethasone dipropionate.
Limit of detection and limit of quantification
LOD and LOQ was determined in accordance with the ICH guidelines. 14 These values were calculated using the slope and standard deviation derived from the calibration curve. (Table 1)
Table 2
Result of regression analysis of luliconazole and beclomethasone dipropionate
Result of regression analysis ofluliconazole and beclomethasone dipropionate
Accuracy
For the accuracy assessment, we conducted experiments using various concentrations of luliconazole and beclomethasone dipropionate. This involved adding a known quantity of luliconazole and beclomethasone dipropionate standards to a fixed amount of the previously analyzed luliconazole and beclomethasone dipropionate sample. The accuracy data was represented as a percentage, reflecting the luliconazole and beclomethasone dipropionate content in the actual samples. We determined the recovered amounts of luliconazole and beclomethasone dipropionate, calculated the % recovery, and computed the %RSD for each concentration level. The summary of these results is provided in Table 2, offering a concise overview of the accuracy assessments conducted.
Table 3
Accuracy data for both drugs
Precision
Repeatability
In compliance with the provided guidelines, a total of five standard solutions, all possessing equivalent concentrations, were meticulously formulated and subsequently introduced into the HPLC instrument. The result was given in Table 3.
Precision (Day to Day variability)
To assess the precision of measurements, intra-day precision involved conducting three replicates of luliconazole and beclomethasone dipropionate peak area measurements on the same day under consistent conditions. Conversely, three separate test runs on different days were used to establish the inter-day precision. The data was then analyzed by calculating the percent relative standard deviation (%RSD), which provides a measure of the variability or consistency in the results. The results were subsequently reported in Table 4.
Table 5
Intermediate precision
Assay
The method we developed was utilized to determine the levels of luliconazole and beclomethasone dipropionate in a topical dosage form. The quantities of these drugs present in the cream were analyzed based on the peak area obtained. The drug content was computed by averaging the results of six separate determinations. The assay results are presented in Table 5, demonstrating the measured drug concentrations in the formulation.
Results
Robustness
Robustness of an analytical method pertains to its capacity to maintain stability and consistency when deliberately subjected to minor variations in method parameters. This assessment offers valuable insights into the method's reliability when used in typical conditions. The evaluation of robustness involves analysing the analytical solution while intentionally adjusting certain physical parameters like flow rate, temperature, and wavelength.15 In this process, one parameter is deliberately altered while the others are held constant, and the resulting chromatogram profile is carefully observed and recorded. This systematic approach aids in quantifying the method's ability to endure slight alterations in these parameters, reinforcing its dependable performance as shown in Table 6.
Table 7
Robustness data for both drugs
Conclusion
The RP-HPLC method proposed in this study was effectively validated according to the criteria outlined by the International Conference on Harmonisation (ICH). The validation encompassed key parameters such as specificity, linearity, system suitability, precision, assay, accuracy, LOD, LOQ, and robustness. The method was characterized by its simplicity, speed, cost-effectiveness, and precision. All validation parameters fell within the acceptable range specified by the guidelines. Notably, no existing chromatographic combination method was identified in the literature for the analysis of luliconazole and beclomethasone dipropionate topical dosage form. Consequently, our efforts were directed towards creating a straightforward, swift, accurate, and precise method of chromatography with reverse phases for the estimation of luliconazole and beclomethasone dipropionate topical formulation. This developed method has been effectively employed in the quality control analysis of luliconazole and beclomethasone dipropionate topical dosage form.
Acknowledgment
Our gratitude extends to the Central Drug Testing Laboratory (CDTL) in Mumbai for generously supplying the samples and standards of luliconazole and beclomethasone dipropionate in topical dosage form. Furthermore, we appreciate their provision of necessary facilities and support for the successful completion of this project.
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