Identification and Determination of Related Substances in Diosmin Bulk Drug and Pharmaceutical Formulations by HPLC and HPLC–MS

Six related substances were detected in diosmin bulk drug substances and products by a newly developed gradient reverse-phase high performance liquid chromatography (RP-HPLC) with UV detection. The chromatographic system consisted of an Intersil Wondasil TM ODS (C 18) column (250 × 4.6 mm; 5 μm). The mobile phase consisted of water/acetic acid 66:6 v/v (solvent A) and methanol (solvent B) using a gradient program at a flow rate of 0.8 mL min−1 with 345 nm detection and an injection volume of 20 μL. In addition, the linearity, quantitation limit (QL), accuracy, selectivity, robustness and precision were determined. Good linearity was obtained over the concentration range 0.5–200 μg mL−1 with the coefficient of determination (r 2) of 0.999. The QL was 0.125 μg mL−1 (relative standard deviation