Rapid isocratic HPLC determination of fipronil, (S)-methoprene, and major related substances using an end-capped monolithic column

In this study, a simple and rapid isocratic reversed-phase high-performance liquid chromatography (HPLC) method was developed. This method allows the simultaneous determination of active substances and their major impurities, fipronil sulfone and cis-methoprene, in spot-on products containing fipronil and (S)-methoprene. Separation was achieved using an end-capped monolithic column, with an isocratic mobile phase composed of acetonitrile and 0.1% formic acid (70:30, v/v) at 1.0 mL/min and UV detection at 245 nm. Compared to conventional particulate columns, this method provides significant advantages, including very low system back-pressure (~13 bar) and a short analysis time (~12 min).The method was validated for selectivity, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), and system suitability. Good linearity was obtained (R² ≥ 0.995), and repeatability (%RSD < 2.0) and recovery values ranged from 95.68% to 99.07%. The method enabled detection of impurities below 0.05%.The environmental impact of the method was evaluated using AGREE (Analytical GREEnness Metric) and GAPI (Green Analytical Procedure Index). The reduced analysis time and low solvent consumption confirmed the method’s compliance with green analytical chemistry (GAC) principles. Following validation, the method was applied to ten commercial products available on the Türkiye market. The active substance content of all samples was within the ±10% specification limits, and the levels of major impurities were found to be below acceptable thresholds.This study proposes a practical, cost-effective, and environmentally friendly HPLC method based on an end-capped monolithic column for routine quality control and impurity profiling.