Development and Validation of a High-Sensitivity HPLC Method for Quantification of Ketoconazole: Ensuring Quality Assurance Through Specific Chromatographic Procedures

Abstract

Analyzing the drug substance serves as a crucial step in verifying the quality of the active pharmaceutical ingredient. This research is aimed at developing an isocratic high-performance liquid chromatography (HPLC) method for the quantitative analysis of ketoconazole in its stable form. The novelty of this work lies in its remarkable specificity and sensitivity, ensuring highly accurate results. Chromatographic separation was performed using an Xterra RP C18 column (250 × 3.0 mm) with a particle size of 5 μm, and detection was carried out with UV at 244 nm. The sequence run time was 8 min, with the average analyte peak eluting at 3.469 min while maintaining a flow rate of 1.0 mL/min. The preparation of both the sample and the mobile phase is rapid and highly economical, significantly reducing the chances of manual error. The analysis duration has been notably reduced to approximately 8 min, with the analyte eluting in under 5 min. This represents a significant improvement in efficiency compared with earlier reports. The method performance was validated following the current International Conference on Harmonization (ICH) guidelines, covering aspects such as specificity, system suitability, limits of detection (LOD) and quantification (LOQ), linearity, accuracy, and precision. Regression analysis revealed a correlation coefficient value of 0.9982. The LOD and LOQ were found to be 10 and 30 μg/mL respectively. The accuracy of the method was evaluated through a recovery study at levels of 80, 100, and 120%, and the values were found to be within the acceptable range (% relative standard deviation [RSD] less than 2%). The RSD for method precision experiments was found to be 1.0%, which complies with the ICH guidelines. This method was successfully developed for the estimation of ketoconazole using a straightforward and effective isocratic technique.