Development and Optimization of a Sensitive LC–MS/MS Method for the Quantification of N‐Nitroso Bisoprolol in Pharmaceutical Formulations

ABSTRACT The trace‐level measurement of N‐Nitroso Bisoprolol, a possible genotoxic impurity (PGI), in pharmaceutical formulations was accomplished by the development, validation, and optimization of a highly sensitive and selective liquid chromatography tandem mass spectrometry (LC–MS/MS) approach. The detection and quantification of nitrosamine impurities at very low levels are essential for patient safety and compliance with international regulatory standards, especially in light of the growing regulatory scrutiny of these impurities due to their propensity to cause cancer. High sensitivity and selectivity are ensured by the method's use of an atmospheric pressure chemical ionization (APCI) source in positive ionization mode and multiple reaction monitoring (MRM) for detection. Using a Zorbax SB Phenyl analytical column (4.6 × 250 mm, 5 µm) and a carefully planned gradient elution procedure, chromatographic separation was accomplished using 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in methanol (mobile phase B). Even in complex matrices, this arrangement provided better peak form, retention, and resolution. The method demonstrated remarkable detection (DL) and quantification (QL) limits of 1.24 and 3.75 ppm, respectively. The method demonstrated excellent linearity ( R 2 = 1.000) over the range from the quantification limit (QL) to 150% of the test concentration. According to ICH Q2(R2) guidelines, validation experiments verified exceptional intra‐ and inter‐day precision (RSD < 5%), accuracy, and robustness.