Development and validation of RP-HPLC method for simultaneous estimation of luliconazole and beclomethasone dipropionate in topical dosage form

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.

Choice of wavelength

A standard solution of 10µg/mL of Luliconazole and Beclomethasone dipropionate was scanned under UV within the 400-200nm range against diluent as a blank. Luliconazole showed maximum absorbance at 290nm and Beclomethasone dipropionate showed maximum absorbance at 242nm.

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.

Preparation of mobile phase (MP)

1% glacial acetic acid (GAA : Acetonitrile (ACN)

(30: 70)

Optimization chromatographic conditions

Column: Hemochrom C18(4.6mm × 250mm × 5µm)

MP: 0.1% Glacial acetic acid: ACN (30:70v/v)

Flow rate: 1ml/min

Wavelength: 242nm, 290nm

Injection: 10μl

Run time: 13 mins

Column temperature: 35ºC

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]

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])

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.

Precision

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].

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.

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].