Determination of Potential Genotoxic NDSRI (Nitrosamine Drug Substance Related Impurity) in Ticagrelor – Antiplatelet Medications: (Q) SAR Assessment, HPLC-ESI-MS/MS Method Development and Validation

Abstract

Regulatory agencies, including the FDA, have identified that commonly used amine-based Active Pharmaceutical Ingredient (API) synthesis processes may lead to the unintentional formation of nitrosamine impurities, which are classified as potentially genotoxic. Although N-nitrosamines are not deliberately introduced during drug manufacturing, they can form as impurities or degradation products under certain processing or storage conditions. The challenge in nitrosamine determination arises from their extremely low acceptable daily intake (ADI) limits, lack of strong chromophores, and the continuously expanding list of controlled nitrosamines, necessitating highly sensitive and selective analytical methods.

This study describes the development and validation of a highly sensitive Liquid Chromatography-Tandem Mass Spectrometry (LC–MS/MS) method for the trace-level quantification of N-nitroso-ticagrelor in ticagrelor API. Chromatographic separation was achieved on a Supelco Ascentis Express C18 column (10 cm × 4.6 mm × 2.7 μm) using a gradient elution with 0.1% v/v formic acid in water and methanol at a flow rate of 0.8 mL/min, with a total run time of 30 minutes. Detection was performed using a triple quadrupole mass spectrometer with electrospray ionization (ESI) in positive mode and multiple reaction monitoring (MRM).

The method was validated in accordance with ICH guidelines. The calibration curve demonstrated excellent linearity with a correlation coefficient (r) of 0.9922. The limits of detection (LOD) and quantification (LOQ), determined using the signal-to-noise approach, were 0.0031 ppm and 0.0093 ppm, respectively. Method recoveries & accuracy were within 70–130%, and all validation parameters met the predefined acceptance criteria. This validated method enables sensitive, accurate, and regulatory-compliant quantification of N-nitroso-ticagrelor, supporting risk assessment and quality control of ticagrelor API.