Development and Validation of an HPLC-UV Method for the Dissolution Studies of 3D-Printed Paracetamol Formulations in Milk-Containing Simulated Gastrointestinal Media

This study presents the development and comprehensive validation of a high-performance liquid chromatography with ultraviolet detection (HPLC-UV) method for quantifying paracetamol (PAR) in milk-containing biorelevant dissolution media. The primary objective was to support dissolution studies of 3D-printed paracetamol formulations, particularly those intended for pediatric use, which often involve complex matrices. The method employed isocratic separation using a mobile phase of 25 mM phosphate buffer (pH 3.0) and methanol (80:20, v/v) at a flow rate of 1 mL/min. A protein precipitation technique was utilized for sample clean-up, and the effectiveness of various precipitation reagents (methanol, acetonitrile, 10% v/v trifluoroacetic acid) was thoroughly investigated to optimize analyte recovery. The matrix effect, a critical consideration for complex biological samples, was assessed by comparing calibration curves in aqueous and matrix-matched media, demonstrating acceptable results (90-110% variation). The method was validated according to ICH guidelines, focusing on accuracy profiles. Key performance indicators included a relative bias between -4.5% and +3.9%, repeatability and intermediate precision (RSD < 2.7% and 3.0%, respectively), a limit of detection (LOD) of 0.02 μg/mL, and a lower limit of quantitation (LLOQ) of 10 μg/mL. The validated method was successfully applied to determine paracetamol release from 3D-printed formulations in a two-stage biorelevant dissolution test, providing a robust tool for assessing drug release kinetics in physiologically relevant conditions.