Simultaneous and trace‐level quantification of four benzene sulfonate potential genotoxic impurities in doxofylline active pharmaceutical ingredients and tablets using high‐performance liquid chromatography with ultraviolet detection

Abstract

AbstractIn the production of doxofylline, the common occurrence of toxic p‐toluene sulfonate generation prompted the development and validation of a method using HPLC with ultraviolet detection (HPLC‐UV). This method is designed for detecting four potential genotoxic impurities (PGIs) present in both doxofylline drug substance and tablets, with a focus on the UV‐absorbing group p‐toluene sulfonate. The four impurities were methyl 4‐methylbenzenesulfonate (PGI‐1), ethyl 4‐methylbenzenesulfonate (PGI‐2), 2‐hydroxyethyl 4‐methylbenzenesulfonate (PGI‐3), and 2‐(4‐methylphenyl)sulfonyloxyethyl 4‐methylbenzenesulfonate (PGI‐4). In this method, chromatographic separation was achieved using a Waters Symmetry C18 column (250 mm × 4.6 mm, 5 μm). The mobile phases consisted of 20% acetonitrile as mobile phase A and pure acetonitrile as mobile phase B, operating in gradient elution mode at a flow rate of 1.0 mL/min. According to the guidelines of the International Conference on Harmonization, it was determined that this method could quantify four PGIs at 0.0225 μg/mL in samples containing 60 mg/mL. The validated approach demonstrated excellent linearity (R2 > 0.999) across the concentration range of 30%–200% (relative to 0.075 μg/mL doxofylline) for the four PGIs. The accuracy of this method for the four PGIs ranged from 94.8% to 100.4%. The reverse‐phase‐HPLC‐UV analytical method developed in this study is characterized by its speed and precision, making it suitable for the sensitive analysis of benzene sulfonate PGIs in doxofylline drug substances and tablets.