Development of a green chemistry based bioanalytical method using response surface methodology to analyze febuxostat and indomethacin in rabbit plasma

This research established an environmentally friendly and sustainable approach to measure Febuxostat and Indomethacin levels in rabbit plasma samples, utilizing isocratic liquid chromatography guided by green analytical chemistry principles and Analytical Quality by Design (AQbD) methodology. Chromatographic separation was performed on an Eclipse Plus C18 column (25 cm x 5 cm,4.6 μm), using a binary mobile phase of ethanol and 50 mM potassium dihydrogen orthophosphate (pH 4.5) in a 66:34 ratio, delivered at 0.8 mL/min for 15 min. Resolution and asymmetry factors were designated Critical Analytical Attributes (CAAs). Control Noise Experimentation (CNX) screening identified flow rate, mobile phase pH, and ethanol concentration as significant contributors to CAAs variability. Subsequent optimization utilizing Central Composite Design (CCD) refined the Critical Method Parameters (CMPs) to ensure optimal performance. Chromatographic analysis revealed Febuxostat and Indomethacin retention times of 4.41 and 7.35 min, respectively. The method’s greenness and analytical quality were assessed using AGREE, ComplexGAPI, RGB, and AMGS tools. Validation studies confirmed linearity (R2: 0.9959 for Febuxostat, 0.9981 for Indomethacin) within 200–4600 ng/mL, alongside successful precision, accuracy, recovery, and stability evaluations at concentrations of 250, 750, 1500, and 3000 ng/mL.