A Robust RP-HPLC Method for Simultaneous Estimation of Paracetamol and Nefopam in Human Plasma: Design of Experiments Approach

A rapid, sensitive, and robust reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the simultaneous estimation of Paracetamol, and Nefopam, in human plasma. The method development was carried out using a Quality by Design (QbD) approach to systematically optimize critical chromatographic parameters for improved resolution and reproducibility. Chromatographic separation was achieved on a C18 column using a mobile phase comprising Acetonitrile and 0.1% orthophosphoric acid in a specified ratio, delivered at a flow rate of 0.7 mL/min, with detection at 255 nm. The retention times for Paracetamol and Nefopam were found to be approximately 6.220 and 7.410 minutes, respectively, indicating excellent peak symmetry and resolution. Method validation was carried out by evaluating key bioanalytical parameters, including selectivity, linearity, accuracy, precision, recovery, matrix effect, and stability under various conditions. The method exhibited a linear response over the concentration range of 10–50 µg/mL for Paracetamol and 1–5 µg/mL for Nefopam with %RSD values within acceptable limits. No significant interference from plasma components was observed at the retention times of the analytes. This validated method is suitable for pharmacokinetic, bioequivalence, and routine therapeutic drug monitoring studies involving these NSAIDs in human plasma.