Analytical Method Development, Validation and Optimization of Fluconazole Drug Using RP- HPLC

Pharmaceutical analysis plays a crucial role in ensuring the quality, safety, and efficacy of bulk drug substances and their formulations. Among the various analytical techniques available, High-Performance Liquid Chromatography (HPLC) has emerged as one of the most reliable and versatile tools for both qualitative and quantitative analysis in the pharmaceutical industry.[1,2] The data generated through HPLC can be either numerical, representing the exact amount of a compound present in a sample, or qualitative, confirming the presence or absence of specific analytes.[3,4] Owing to its high sensitivity, accuracy, and reproducibility, HPLC is employed at every stage of drug development from the analysis of raw materials to in-process controls and final product testing.[5,6] The specific objective of each analysis depends on the nature of the sample and the stage of pharmaceutical development. Therefore, a clear understanding of the fundamental principles of chromatography is essential to comprehend the operation and applications of HPLC in pharmaceutical analysis. In this research work, Fluconazole was selected as the model drug because it is a widely used triazole antifungal agent employed in the treatment of various systemic and superficial fungal infections. Despite its extensive clinical use, the available analytical methods for Fluconazole are often reported in combination with other drugs or involve complex mobile phase systems containing buffers or multiple solvents, which make them less suitable for routine analysis. Several analytical methods for the estimation of Fluconazole have been previously reported in the literature; however, most of these methods involve complex mobile phase compositions, buffer systems, or drug combinations, which limit their simplicity and routine applicability. Therefore, the present study aimed to develop a simple, reliable, and eco-friendly RP-HPLC method for the quantitative estimation of Fluconazole as a single drug component. Multiple optimization trials were performed using different ratios of Water and Acetonitrile as the mobile phase to achieve a sharp, symmetrical peak with an acceptable retention time and minimal tailing. The use of a this mobile phase not only reduced the organic solvent consumption but also made the method more environmentally friendly, cost-effective, and suitable for routine quality control analysis. Fluconazole, α-(2.4-diflurofenil)-α-(1H-triazol-1- methyl)-1H-1,2,4-triazol-1-ethano an antifungal medication, was discovered and developed by Pfizer. [7,8] Fluconazole is a first-generation triazole antifungal medication. It differs from earlier azole antifungals (such as ketoconazole) in that its structure contains a triazole ring instead of an imidazole ring. [9,10] The presence of a triazole and a difluoro phenyl produces potent antifungal activity but replacement of the usual imidazole group by triazole leads to improved selectivity.[11,12] Fluconazole is much less lipophilic than other azole antifungals and this leads to excellent penetration throughout the body, low protein-binding and water-solubility.[13,14] Fluconazole, was chosen for development on the basis of an optimal combination of antifungal efficacy, pharmacokinetic characteristics, aqueous solubility, and safety profile.[15]

Figure 1: Structure Of fluconazole [16]

Fluconazole is a very selective inhibitor of fungal CYTOCHROME P450 dependent enzyme lanosterol 14-α-demethylase. This enzyme normally works to convert lanosterol to ergosterol, which is necessary for fungal cell wall synthesis. [17]

MATERIALS AND METHOD

• Chemicals and reagents

1. API used in Research Work

Fluconazole Drug Trials (Sample and Standard) was provided by R&D Department of Axiom Analytical Services, Rau, Indore which was manufactured by Rusan Pharma Ltd., Pithampur, Indore.

2. Chemical used

Methanol (HPLC grade) and Acetonitrile (HPLC grade) from Advent Chembio Pvt. Ltd. and Water (HPLC grade) from Rankem Laboratory Chemicals.

3. Major Instrument Used

• HPLC method Development and Validation:

Mobile phase optimization: Initially, the mobile phase tried was Water: Acetronitrile and then one trial with Water and Methanol   with various combinations of then varying proportions. Finally, the mobile phase was optimized to Water and Acetronitrile in proportion 60: 40 v/v respectively.

Wave length selection: UV spectrum of 10 µg / ml Fluconazole in diluents (mobile phase composition) was recorded by scanning in the range of 200nm to 400nm. From the UV spectrum wavelength selected as 260nm.

Preparation of Mobile Phase: Accurately measured of 60 ml volume of water and 40ml volumes of Acetronitrile. were mixed and degassed in an ultrasonic water bath for 10 minutes and then filtered through 0.45 µ filter.

Standard solution preparation: About 10 mg of Fluconazole drug was weighed using weighing balance. The 10mg drug was transferred into clean 100 ml volumetric flask dissolved the drug with H?O (60) & ACN (40) (diluents) and after dissolving, made up the volume (100ml) of the volumetric flask up to the mark, sonicate gently to dissolve completely. Labelled flask as Fluconazole standard solution of 100ppm.

Sample solution preparation: About 10 mg of pure Fluconazole drug was weighed using analytical balance. The 10mg drug was transferred into clean 100 ml volumetric flask dissolved the drug with H?O (60) & ACN (40) (diluents) and after dissolving, made up the volume (100ml) of the volumetric flask up to the mark sonicate gently to dissolve completely. Labelled flask as Fluconazole sample solution of 100ppm.

Method development: The method development for Fluconazole by Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) was carried out with the aim of achieving an efficient, precise, and eco-friendly analytical procedure that provided a sharp, symmetrical peak with reduced tailing factor, shorter retention time, and satisfactory theoretical plates. Various chromatographic conditions were systematically optimized by varying the mobile phase composition, flow rate, wavelength, and column type. Several mobile phase combinations of Water and Acetonitrile (ACN) were evaluated in different ratios ranging from 70:30 to 60:40 (v/v) to obtain optimum separation and peak symmetry. Initial trials did not meet the desired chromatographic performance as the peaks exhibited higher tailing and longer retention times. Subsequently, the mobile phase composition was optimized to Water: Acetonitrile (60:40 v/v), which provided a sharp, symmetrical, and well-resolved peak under the acceptance criteria. The flow rate was adjusted at 1.5ml/min, which provides good peak shape, retention time, tailing factor and run time. Wavelength was chosen at 260nm at this wavelength Fluconazole shows strong absorbance. Injection volumn was adjusted at 10µl and run time was 10 minutes. Under those conditions the Fluconazole eluted at 2.231 minutes.

5. Chromatographic Condition for Method Validation:

Figure No. 2: Optimized Chromatogram of Fluconazole

• Method validation: The method validation was conducted according with International Conference on Harmonization the guidelines. The methods were accuracy, precision, linearity, specificity, and system suitability all were verified. [18]
• System suitability: Standard solution of drug was prepared using water and acetonitrile as solvent (ppm). Blank sample and standard solution were injected and the run was performed. Parameters such as retention time, tailing factor, plate count, peak area was determined. The measured results were compared against the pre-defined limits. By inject blank, standard solution and parameters such as retention time, tailing factor, plate count, peak area was found in acceptance criteria, it concluded that system was suitable for further analysis. [19]

• Tailing factor < 2.0
• Theoretical plates > 2000
• Should be consistent (±2%)

• Specificity: In order to verify specificity by inject blank, sample solution and standard solution(100ppm) to check whether there is no interference of blank chromatogram in retention time of Fluconazole and that confirmed that the method is specified for fluconazole drug. [20]
• Linearity: Along with the blank and standard, six different concentrations of the standard solution (120–900μg/mL) of the target concentration were injected to ensure linearity. After the calibration curve was established between concentrations and peak regions, regression analysis was performed. A linear technique was demonstrated if the correlation coefficient fell within the permitted range.

     Correlation coefficient (R²) ≥ 0.999

• Precision: Method were assessed by preparing and injecting six individual sample solution on identical analyte condition and record peak area or region of all injection and calculate the assay, %RSD if all are within the acceptance criteria than the method was precised. [21]
• Accuracy: By injecting blank, standard and sample solution in triplicate time at different concentration of 50%,150% and 100%. Calculate peak area that how much amount of drug or API were recovered at each level. Determined mean recovery % and % RSD for each level and if mean recovery % and % RSD were within acceptance limit than the method was accurate. [22]

RESULTS AND DISCUSSION:

• System suitability: Inject blank, standard solution and observed the chromatograph.

Table No 1: System suitability and precision

Figure No.3: System suitability chromatogram

• Specificity: Inject black, standard, sample solution of 100ppm and observed the chromatograph.

Table No. 2: Specificity of Fluconazole

• Linearity: Inject blank, standard of 6 replicates of different concentration and observed the data.

Table No. 3: Specificity of Fluconazole

Fig No. 4: Linearity of Fluconazole Graph

• Precision: Inject blank, standard, six samples

Table No. 3:    Precision of Fluconazole

Fig. No. 5: Precision chromatogram

• Accuracy: Inject blank, standard, 3 replicate of each 50%sample, 100% sample, 150% sample and   observed the chromatograph.

Table No. 4 Accuracy of Fluconazole

Figure No. 6: Standard Chromatogram 50%

Figure No.7: Standard Chromatogram 150%