Development and Validation of a Highly Sensitive LC–MS/MS Method for Ultra‐Trace Quantification of N‐Nitroso Desmethyl Venlafaxine in Venlafaxine Drug Substance

ABSTRACT N‐nitrosamine impurity control is rigorously required by regulators owing to their latent oncogenic risks. In this study, a rapid and highly sensitive liquid chromatography coupled triple quadrupole mass spectrometry method was developed for the trace‐level quantification of the N‐nitroso desmethyl Venlafaxine (N‐NDVLX) in the Serotonin–Norepinephrine Reuptake Inhibitor antidepressant Venlafaxine (VLX) drug substance. As per the carcinogenicity potency categorization approach, N‐NDVLX is categorized as category 1, with an acceptable intake limit of 18 ng/day. Chromatographic separation was accomplished on an Agilent Eclipse EC‐C18 column (150 × 4.6 mm, 2.7 µm particle size) using 0.1% formic acid aqueous solution and acetonitrile under gradient mode of elution at a 0.4 mL/min flow rate. Detection was made using triple quadrupole mass spectrometry with electrospray ionization in multiple reaction monitoring mode, employing transitions m/z 293.15 > 121.02 (quantifier) and 293.15 > 202.12 (qualifier). The method was validated following ICH Q2(R2) guidelines and established excellent sensitivity, specificity, and robustness. This method is highly practical for N‐NDVLX routine testing, ensuring regulatory compliance through highly sensitive and reproducible trace‐level quantification. Minimizing this potentially genotoxic impurity directly supports patient safety while enhancing the chemical quality and purity profile of the VLX drug substance.