A Highly Sensitive Liquid Chromatography–Triple Quadrupole Mass Spectrometric Method for the Simultaneous Trace‐Level Quantification of Trofinetide N‐Nitrosamine Impurities in Trofinetide Drug Substance

Abstract

Regulatory authorities worldwide mandate stringent monitoring and control of N‐nitrosamine drug substance‐related impurities due to their potential carcinogenic risk, in order to ensure the safety, quality, and efficacy of pharmaceutical products throughout their lifecycle. In this context, the present study investigates the simultaneous quantification of N‐nitrosamine impurities in the neurodevelopmental disorder agent trofinetide (TND). The primary objective was to develop a rapid, accurate, and highly sensitive liquid chromatography–triple quadrupole mass spectrometry method for the simultaneous trace‐level quantification of (S)‐2‐methyl‐1‐nitrosopyrrolidine‐2‐carboxylic acid (N‐NTND‐1) and methyl (S)‐2‐methyl‐1‐nitrosopyrrolidine‐2‐carboxylate (N‐NTND‐2) in its drug substance. Based on the Carcinogenic Potency Categorization Approach, both N‐nitrosamines are classified under potency category 4, with acceptable intake limits of 1500 ng/day. Chromatographic separation was achieved using an Agilent Eclipse C18 column (150 mm × 4.6 mm, 2.7 µm), employing 0.1% formic acid and acetonitrile as mobile phases in gradient elution at a flow rate of 0.4 mL/min. The simultaneous quantification was performed using multiple reaction monitoring (MRM) technique with electrospray ionization in positive ion mode. The optimized mass transitions were m / z 158.91 > 67.04 (quantifier) and 158.917 > 42.977 (qualifier) for N‐NTND‐1, and m / z 173.02 > 67.04 (quantifier) and 173.02 > 84.14 (qualifier) for N‐NTND‐2. The developed method was validated in accordance with ICH Q2(R2) guidelines, with all validation parameters meeting predefined acceptance criteria. The method was successfully applied to the analysis of commercial TND batch samples, demonstrating high sensitivity.