Synthesis, Method Validation, and Mechanistic Analysis of Three Mutagenic Impurities in Apremilast

ABSTRACT Genotoxic impurities (GTIs) are highly potent mutagenic substances that typically require control to low ppm levels in pharmaceuticals. A novel limit test method (single impurity ≤ 25 ppm) based on high‐performance liquid chromatography‐tandem mass spectrometry was established and partially validated for the determination of three GTIs in Apremilast. Chromatographic separation was achieved on an Agilent SB C18 column using gradient elution with mobile phase A consisting of 1 mmol/L ammonium acetate solution (containing 0.1% formic acid) and mobile phase B of acetonitrile. The three impurities were detected in electrospray positive ionization mode using time‐of‐flight mass spectrometry at the following mass‐to‐charge ratios: m/z 466.13 ± 0.02 Da (APST‐ZZ4), m/z 581.17 ± 0.02 Da (APST‐ZZ10), and m/z 611.14 ± 0.02 Da (APST‐ZZ11). The developed method demonstrated good specificity and high sensitivity, with limits of detection ranging from 0.50 to 1.00 ng/mL. The genotoxicity of these impurities was evaluated using two (quantitative) structure‐activity relationship prediction methodologies, indicating that APST‐ZZ4, APST‐ZZ10, and APST‐ZZ11 possess significant mutagenic risk. Furthermore, the possible mechanisms of action were analyzed and proposed. This study provides instructive insights for the analysis and genotoxicity research of GTIs, and offers technical support for drug regulatory supervision.