Development and Validation of a Highly Sensitive Liquid Chromatography–Tandem Mass Spectrometry Method for the Quantitative Determination of Mutagenic N‐ Nitroso Mefenamic Acid in Pharmaceutical Formulations

Abstract

N‐ Nitrosamines and drug substance‐related nitroso impurities represent a major regulatory concern in modern pharmaceutical development due to their classification as human mutagens. Regulatory frameworks emphasize structural evaluation, establishment of acceptable intake limits, and the implementation of highly sensitive analytical methods to ensure patient safety. Mefenamic acid (MAA), an anthranilic acid derivative and widely used nonsteroidal anti‐inflammatory drug, functions by inhibiting prostaglandin synthesis to relieve pain, fever, and inflammation. However, its molecular structure contains a secondary amine, predisposing it to the formation of N‐ nitroso MAA ( N‐ NMAA), a mutagenic impurity of significant toxicological relevance. This study presents a comprehensive structural investigation and toxicological assessment of N‐ NMAA, classified under Carcinogenic Potency Category 5 with an acceptable daily intake limit of 1500 ng. To address regulatory requirements, a highly sensitive liquid chromatography–tandem mass spectrometry method was developed and validated for trace‐level quantification of this impurity in drug formulations. The method utilizes a Poroshell Stable Bond‐C18 column, with a gradient mobile phase of ammonium formate buffer and acetonitrile at 0.6 mL/min. Mass spectrometric detection was achieved in electrospray ionization mode, monitoring precursor and product ions at m/z 269.0 and 239.1. This validated method ensures reliable detection and control of mutagenic impurities in MAA formulations.