A sustainable RP-HPLC method for simultaneously estimating aceclofenac, paracetamol, and serratiopeptidase with AQbD optimization

• Simultaneous Estimation: A novel RP-HPLC method was developed for AFC, PCM, and SPD. • Green and White Analytical Chemistry: The method emphasizes sustainability and reduces revalidation needs. • Optimized Separation: Using an Agilent Inertsil ODS column retention times are 3.5, 5.2, and 8.9 min. • Validation Standards: The method shows %RSD values below 2%, ensuring reliable results. • Sustainability in Analytical Methods: Integrating AQbD and WAC, it aligns with eco-friendly trends. This study focused on developing and validating a novel method for the simultaneous estimation of aceclofenac (AFC), paracetamol (PCM), and serratiopeptidase (SPD) using reverse phase high-performance liquid chromatography (RP-HPLC). The method was designed based on Analytical Quality by Design principles, incorporating green and white analytical chemistry (WAC) to ensure environmental sustainability and analytical efficiency. The HPLC analysis was performed using an Agilent 1220 Infinity II system equipped with a PDA detector, binary solvent pump, and automatic injector. Separation was achieved on an Agilent Inertsil ODS 3 column (250 mm × 4.6 mm, 5 μm particle size) with a mobile phase of ethanol and a 10 mM phosphate buffer (pH 3), adjusted with 1% orthophosphoric acid. A gradient elution method was employed with a flow rate of 0.8 mL/min and detection at 230 nm. The retention times for AFC, PCM, and SPD were found to be 3.5, 5.2, and 8.9 min, respectively. Method validation confirmed accuracy and precision, with relative standard deviation (RSD) values consistently below 2%, and a linearity range that met acceptable analytical standards. The integration of green and white analytical chemistry principles enhanced the method's sustainability, reducing environmental impact while ensuring minimal revalidation requirements. By adopting WAC, the method demonstrated simplicity, stability, and a strong alignment with sustainable chemistry practices. This method serves as an innovative and environmentally friendly approach for the accurate analysis of pharmaceutical compounds.