Method Development and Validation of Sustainable RP-HPLC Analytical Methods for Clonidine Hydrochloride Using AQBD Optimization

Abstract

A sustainable and robust reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated for the estimation of clonidine hydrochloride using green analytical principles with Analytical Quality by Design (AQbD) optimization. Chromatographic separation was achieved on a ZORBAX Eclipse Plus C18 column (250 × 4.6 mm, 5 µm) using an Agilent 1220 Infinity II HPLC system equipped with a binary pump, autosampler, and PDA detector. The mobile phase consisted of water and phosphate buffer (50:50 v/v), adjusted to pH 5, and was eluted in isocratic mode at a flow rate of 1 mL/min. The detection wavelength was set at 245 nm with an injection volume of 10 µL, and the column oven temperature was maintained at 35°C.The developed method possessed good linearity over the concentration range of 5–15 µg/mL with a regression equation of y = 372.84x + 145.62 and a correlation coefficient (R²) of 0.999. The limit of detection and limit of quantification were found to be 0.52 µg/mL and 1.58 µg/mL, respectively. The method exhibited accuracy (98.7–100%) and high precision, with %RSD values of 0.64 (interday) and 0.49 (intraday). The developed method is simple, precise, accurate, and environmentally considerate, aligning with sustainable analytical chemistry principles. It is suitable for routine quality control analysis of clonidine hydrochloride in pharmaceutical formulations.