Development of Robust RP-HPLC Method for Posaconazole Assay in Liposomal Formulation using QbD and ICH-Guided Validation with Forced Degradation Analysis

Abstract

Posaconazole, an antifungal for invasive infections, requires liposomal formulation to overcome poor solubility and bioavailability, making robust analytical methods essential to ensure quality despite interference from excipients. The aim was to develop and validate a reverse-phase HPLC method for the quantification of posaconazole in a liposomal formulation, utilizing Design Expert software. This method was developed to ensure consistency and reliability as per the ICH guidelines. Chromatographic separation was carried out using a reverse-phase HPLC system assembled with a C₁₈ (ODS) column (150 × 4.6 mm, 3.5 µm). A mixture of methanol and buffer in a 70:30 (v/v) ratio served as the mobile phase, with a flow rate maintained at 1 mL/min. A Quality by design (QbD) approach was employed to optimize key chromatographic conditions, including the ratio of the mobile phases, pH and rate of mobile phase flow. The validation of the developed method was done based on linearity, accuracy, precision, sensitivity, specificity and robustness. The optimized RP-HPLC method showed a linear response with a correlation coefficient (r²) of 0.9994 across the concentration range of 800-1200 µg/mL, ensuring accurate quantification of posaconazole in liposomal formulation. Precision studies demonstrated low relative standard deviations (RSD), with intra-day precision of 0.12% and inter-day precision ranging from 0.36% to 0.39%. The accuracy of the method was 98.4% to 99.4%, confirming its reliability for routine use. Ruggedness testing yielded consistent results from two analysts, with RSD values of 1.38% and 1.42%, indicating the reliability and robustness of the method. Specificity testing confirmed that the method can accurately quantify posaconazole in the presence of formulation excipients, with no interference observed. Furthermore, forced degradation stability studies revealed the stability-indicating ability of the method in terms of a clear distinction between the drug and subsequent degradation products. Based on results, this validated method provides a reliable tool for ensuring the consistent quality and therapeutic efficacy of posaconazole liposomal formulation.