Method Development And Validation Of Luliconazole By UHPLC In Bulk And Topical Dosage Form

Luliconazole, also known by the commercial name Luzu, is an imidazole-based antifungal drug. It is recommended as a 1% topical cream to treat ringworm, jock itch, and athlete's foot brought on by dermatophytes like Trichophyton rubrum, Microsporum gypseum and Epidermophyton floccosum. The imidazole class includes it. Though it's unclear exactly how they work to combat dermatophytes^[1,2]. The azoles obstruct the ergosterol production pathway's lansterol 14?-demethylase, which prevents lanosterol from being converted to ergosterol. An inadequate quantity of ergosterol causes intracellular 14? methyl sterols to accumulate, which inhibits growth and ultimately results in mortality. It exhibits activity against several types of fungi, including Aspergillus, Trichophyton, Tinea, and Epidermatophytes in particular^[3,4]. Various analytical techniques, including UV spectrophotometry^[5,6], liquid chromatography^[7], TLC^[8], HPTLC^[8,9] , RP-HPLC^[10,11,12], RP-UFLC^[13] and other techniques^[14,15], ingredients and experimental design were released for the purpose of evaluating luliconazole in biological fluids and pharmaceutical formulations. In the current study, we have created a straightforward UHPLC method for quantification of luliconazole in pharmaceutical dosage form and bulk that indicates economic stability and accuracy. The procedure was verified in accordance with ICH recommendations. A new antifungal medication called LCZ was introduced by Ranbaxy Laboratories Ltd. in India. Originally, the molecule was screened from active ingredients associated with the powerful antidermatophytic medication lanoconazole. Luliconazole (LCZ) is a medication that is highly effective against dermatophytes and a member of the imidazole class of drugs. It has a broad range of antifungal activity. Luliconazole is chemically, (2E)-2-[(4R)-4-(2, 4-dichlorophenyl)-1, 3-dithiolan-2-ylidene]-2-imidazol-1-yl-acetonitrile (C₁₄H₉Cl₂N₃S₂) with molecular weight 354.28. It is used as an antifungal agent. The double bond next to the dithiolane molecule in the R-enantiomer of luliconazole is arranged in an E configuration, and it has just one chiral center^[13]. It has been discovered that luliconazole has a wide range of antifungal action against pathogenic fungi, particularly dermatophytes. 1% creams and solutions are mostly used to treat superficial infections including candidiasis, pytyriasis versicolor, and dermatophytosis. It is recommended for the treatment of dermatophytes like Trichophyton robrum, Microsporum gypseum, and Epidemophyton floccosum that cause ringworms, jock itch, and athlete's foot. An azole antifungal called luliconazole cream is prescribed for the topical management of interdigital tinea pedis, tinea crusis, and tinea corporis. I have created a straightforward, precise, and cost-effective UHPLC method in this study to quantify luliconazole in topical and bulk dose forms. Method validation followed ICH norms^[16].

UHPLC is a technology that has the potential to reduce operation costs while also achieving greater resolution, faster analytical times, and increased separation efficiency. Ultra high-pressure liquid chromatography, also known as performance liquid chromatography, involves the use of sub-2 ?m particle-packed columns in combination with a system capable of withstanding extremely high pressures (up to 1500 bar). This chromatographic method aims to boost peak capacity and/or sample throughput ^[17].                                

       
           
       
Fig.1: Structure of luliconazole ^[13]

MATERIAL AND METHODS

Selection of solvent:

Methanol was selected as the solvent for dissolving Luliconazole.

Selection of analytical wavelength:

Methanol as a blank and Luliconazole standard solution (20 PPM) was scanned from 400 nm to 200 nm. Absorption maxima was determined for drug.

UHPLC Method Development and Optimization of chromatographic conditions:

Preparation of standard stock solution for Chromatographic development: Luliconazole Standard stock solution was prepared by transferring 10 mg Luliconazole into a 20 mL clean and dried volumetric flask, added about 15 mL of Methanol to dissolve it completely and made volume up to the mark with methanol. (500 PPM). Further diluted 2 ml of stock solution to 10 mL with Mobile phase. (100 PPM). The standard solutions of Luliconazole was used for UHPLC Method development.                                                              Selection of detection wavelength for UHPLC method development: Analytical wavelength for the examination was selected from the wavelength of maximum absorption from the spectrophotometric analysis and it was 296 nm.

Selection of mobile phase: Mobile phase was prepared by mixing the MEOH: Water  in the ratio of 90:10% v/v and the mixture degasified by vacuum filtration using 0.45? filter and sonication. Optimization can be started only after reasonable chromatogram has been obtained. Reasonable chromatogram means that more or less symmetrical peak on the chromatogram after detection. By slight change in mobile phase composition, the position of peak can be predicted within a range of investigated changes. An optimized chromatogram was the one where peak of Luliconazole was symmetrical and well separated within 8 minute of run time. The Mobile phase was selected on the basis of best separation, theoretical plate and tailing factor, peak shape, peak stability etc. Numbers of trials were taken for selection of mobile phase. Initially different proportions of methanol-water, Methanol-ACN were tried.  Finally, a Methanol : water was used in ratio of 90:10v/v.

Optimization of chromatographic conditions: optimized chromatography which is as follows:

Mode: Isocratic

Detector: U.V. Detector

Column Name: Waters cortex C18,

Column Dimension: (150 mm X 4.6 mm i.d.) 2.7 ?m.

Column Oven temperature: 40°C

Injection Volume: 20 ?l

Wavelength: 296 nm

Mobile phase: Methanol : Water (90:10)

Flow Rate: 1.0 ml/min.

Run time: 8 minutes

VALIDATION OF UHPLC METHOD ^[6,16]

The developed chromatographic method was validated for system suitability, linearity, range, accuracy, precision, LOD-LOQ and robustness parameters According to Q2A (R1) ICH guidelines. The developed method for estimation of Luliconazole was validated in accordance with ICH guidelines for following parameters.

Linearity And Range: The linearity was determined by analyzing 5 independent levels of calibration curve in the range of 1-15µg/ml. absorbance of each solution against methanol was recorded at 296nm.The calibration curve of absorbance vs conc. Was plotted and correlation co-efficient and regression line equation for luliconazole were determined. Linearity was performed from 10% to 150% of working concentration. Linear regression data as well as calibration curve were shown in table no.1 and 2 under result and discussion section.

Precision: intra-day precision was determined by analyzing luliconazole at 6 different samples of the same day and inter-day precision was determined by analyzing luliconazole at 6 different samples on different days and %RSD was calculated.

The values of  % relative standard deviation (% RSD) for both the parameters are shown in table no.3 under result and discussion. Standard deviation or relative standard deviation are commonly used to express the precision of an analytical process.

Accuracy (% Recovery) : By determining the percentage recovery of luliconazole from the topical dosage form, the method's accuracy was assessed. Recovery studies were carried out by applying the method to topical dosage form containing Luliconazole at 50, 100 and 150% levels. At each level three determinations were carried out and the results are shown in table no.5  under result and discussion. The amount of luliconazole was calculated at each level and % recovery were computed. Accuracy will be conducted in the range from 50 % to 150 % of working concentration. Each accuracy level's solution was created in triplicate. Calculated % Recovery for each sample, Mean % recovery for each level and overall recovery and also calculated % RSD for each level and % RSD for overall recovery.

Robustness: Robustness of the optimized method was studied by changing column wavelength (±3 nm), temperature (±2°C), and flow rate (±10%) during analysis. The sample was injected in triplicate for every condition and % RSD was calculated for each condition is shown in table no. 4 under result and discussion section.

Limit of Detection (LOD) And Limit of Quantitation (LOQ): The LOD and LOQ were estimated from the set of 5 calibration curves used to determine method of linearity. Calibration curves were plotted for each set. These calculations were used to determine LOD and LOQ based on the y-intercept standard deviation and the calibration curve's average slope was used to calculate LOD And LOQ using following formulae.

LOD= 3.3×avg SD/ slope

LOQ=10×avg SD/ slope

Where, SD is the standard deviation of y-intercepts of the calibration curves; S is the mean slope of six calibration curves.

RESULT AND DISCUSSION

UV spectrophotometric Analysis

       
           
       
Fig.2: UV Spectrum of Luliconazole

Observation: The standard solution was scanned between 200 nm to 400 nm. Wavelength of maximum absorption was determined for drug. Luliconazole showed maximum absorbance at 296 nm. 296 nm considered as an analytical wavelength for further determination as it reported in research article.

Linearity And Range

Calibration curve for luliconazole

       
           
       
Fig. 3: Calibration Curve of LCZ

Working standard Solution: 10?g/ml concentration. UV range scanned: 200–400 nm using methanol as blank. The value of ? max was found to be 296 nm.

       
           
       
Table 2: Summary UHPLC linearity of Luliconazole.

The respective linear equation for Luliconazole was:

Y = M X +   C

Y = 994134.8835 x + 32001.5132

 Where, x = concentration of Analyte in µg/mL, y = area of peak, M = Slope, C= Intercept

Precision Studies: Inter-day precision studies were performed using 6 sample. The method is precise as the % RSD values (Table) were found within an acceptable limit.

Robustness of analytical method: One factor was change at one time to estimate the effect. Robustness of method was evaluated for Luliconazole. Irrelevant change in peak area and less variability in retention time were observed. The results of robustness studies are shown in (Table No.4) Robustness parameters were also found satisfactory; hence the analytical method would be concluded.

Overall Recovery: 99.41 %