Quantitation of 7 N-nitrosamines in Monoclonal Antibody (mAb) Formulations Using LC-MS/MS

User Benefits

- An LC-MS/MS method for the low-level quantitation of N-nitrosamines in mAb formulations - A quick, easy and reliable method for complex biological formulation such as mAb

Introduction

N-nitrosamine impurities have long been a concern for pharmaceutical manufacturers, and their testing has become increasingly critical. Some of these compounds are listed as Class 1 mutagens in ICH M7 and have been monitored extensively in since 2018. However, following the conclusion of the review under Article 5(3), the Committee for Medicinal Products for Human Use (CHMP) of European Medicines Agency (EMA) considered that there is also a risk of presence of N-nitrosamines in biological medicinal products as well. This is for the biological medicines with the following risk factors namely biologicals containing chemically synthesized fragments, nitrosating reagents in processes and contamination from packaging, storage and such due to nitrocellulose. Monoclonal antibodies (mAbs) are large, complex biomolecules that can interfere with LC-MS/MS analysis by clogging the chromatography column and contaminating the MS system. To address this, a quick, reliable, and robust sample preparation method was used for low-level quantitation. This application news presents a validated LC-MS/MS procedure for quantifying seven N-nitrosamines in mAb formulations using the Nexera X3 UHPLC coupled with the LCMS-8060NX Triple Quadrupole Mass Spectrometer. The seven N-nitrosamines and four labelled internal standards (IS) include N-nitroso-N-methyl-4-aminobutyric acid (NMBA); N-nitroso-dimethylamine (NDMA); N-nitroso-diethylamine (NDEA); N-nitroso-ethyl-isopropylamine (NEIPA), N-nitroso-diisopropylamine (NDIPA); N-nitroso-dipropylamine (NDPA), N-nitroso-dibutylamine (NDBA); N-Nitroso-N-methyl-4-aminobutyric Acid-d3 (NMBA-d3), N-Nitrosodimethylamine-13C2-d6 (NDMA-13C2 d6), N-Nitrosodiethylamine-d10 (NDEA-d10) and N-Nitroso-di-n-butylamine-d18 (NDBA-d18). The above listed compounds cover the scope of regulatory methods from USP, USFDA and EDQM for N-nitrosamines testing.

August 7, 2025 GMT