Development and validation of a high-performance liquid chromatography method for the quantification of nystatin in a lipid nanoparticle system

Abstract

This study aims to establish and validate an accurate high-performance liquid chromatography (HPLC) method for the quantification of nystatin (NYS) in nanostructured lipid carrier (NLC) systems, thereby addressing the limitations regarding the poor solubility and low permeability of this Biopharmaceutics Classification System (BCS) class IV drug. Methods: The analysis was performed using a Waters e2695 HPLC system equipped with a C18 column (250x4.6 mm, 5 µm). The optimised mobile phase consisted of methanol, acetonitrile, and 0.05 M ammonium acetate buffer (55:15:30, v/v/v), delivered at a flow rate of 1.0 ml/min with UV detection at 305 nm. The sample preparation protocol utilised 5% DMSO (Dimethyl Sulfoxide) as an intermediate solvent to disrupt the lipid matrix. Results: The method demonstrated high system suitability, with Relative Standard Deviations (RSD) for retention time and peak area of less than 2%. Linearity was established within the concentration range of 20-200 IU/ml, yielding a correlation coefficient (R2 ) of 0.9996. The Limit of Detection (LOD) and Limit of Quantification (LOQ) values indicated high sensitivity. The method’s accuracy was confirmed with recovery rates ranging from 98.18 to 101.38%. Both repeatability and intermediate precision met the acceptance criteria with RSD values <2.0%. Notably, the method demonstrated absolute specificity with no interference from the complex lipid matrix. Conclusion: The developed HPLC procedure is simple, reliable, and accurate, making it suitable for quality control and the evaluation of drug loading efficiency in nystatin-loaded lipid nanoparticle systems.