Development and validation of RP-HPLC for the Determination of Caspofungin in the Ufasomes Dispersion, Gel and Rat Plasma

Abstract

Caspofungin is a lipopeptide echinocandin antifungal and its mechanism involves inhibition of (1,3)-β-D-glucan synthase, disrupting fungal cell wall integrity. This study presents a validated RP-HPLC method for detecting and quantifying CSP in ufasomes dispersion and rat plasma, providing a reliable and reproducible approach for pharmaceutical analysis. The method employed a C18 column with a mobile phase of phosphate buffer (pH 2.5) and acetonitrile (65:35, v/v), with fluorescence detection (excitation/emission 224 nm). The caspofungin diacetate (CSP) standards and samples were prepared in methanol and plasma and filtered prior to analysis. Method validation followed ICH Q2 and FDA guidelines, assessing linearity, specificity, precision, accuracy, LOD, LOQ, and matrix effects. The method showed excellent linearity in methanol and plasma (R² = 0.9999), high specificity against excipients, recovery of 97.9% with RSD 1.21 0.36%, and low detection limits (LOD = 1 μg/mL, LOQ = 3 μg/mL). Matrix effects caused longer retention times in plasma compared to ufasomes dispersion, but peak resolution remained clear. Overall, the developed HPLC method is sensitive, accurate, and robust, suitable for routine quality control and research applications of CSP in pharmaceutical formulations.