Determination of Potential Genotoxic Impurities in Esomeprazole Magnesium by HPLC–MS/MS

Esomeprazole magnesium (EMO) is the S -enantiomer of omeprazole. As a second-generation proton pump inhibitor, it is more effective than omeprazole in inhibiting gastric acid secretion. Therefore, it is of great necessity to establish a quality control method to support its pharmaceutical development. The starting materials for the preparation of EMO may contain six potential genotoxic impurities (PGIs), designated as RC16, RC17, RC18, RC20, RC21, and RC23. These impurities have similar chemical structures, all containing benzene rings or pyridine rings with small differences in substituents, which leads to similar retention behaviors in the chromatographic column, and peak overlap is likely to occur during HPLC analysis, making it impossible to accurately determine the content of impurities. To enhance the quality control, this study established a sensitive and specific HPLC–MS/MS method. Utilizing electrospray ionization (ESI) in positive multiple reaction monitoring (MRM) mode, this method was used to determine the trace amounts of six PGIs in EMO raw material. On a 40 °C Inertsil ODS-SP C18 chromatographic column (4.6 × 150 mm, 5 μm), the six PGIs were well separated in an equilibrated elution mode at a flow rate of 0.5 mL min⁻ 1 . 0.5% formic acid in water and methanol were used as the mobile phase A and B, respectively. The method was rigorously validated in terms of precision, accuracy, limit of detection, limit of quantification, linear range, and robustness, and with all parameters meeting pre-defined acceptance criteria. Graphical