Simultaneous determination of 13 nitrosamine impurities in biological medicines using salting-out liquid-liquid extraction coupled with liquid chromatography tandem mass spectrometry.

Nitrosamine impurities are being detected in various pharmaceutical products recently. However, no analytical method is provided for biopharmaceuticals. In present work, a salting-out liquid-liquid extraction (SALLE) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for quantification of thirteen nitrosamine contaminations in antibody drugs. The method showed excellent linearity over the range of 0.5-5.0 μg/L with LOQ (limit of quantitation) of 0.5 μg/L for targeted nitrosamines. The method was demonstrated to be accurate (recovery in a range of 75.4-114.7 %) and precise (RSD ≤ 13.2 %) for all nitrosamines using spiked samples. Especially, we found that the satisfactory recoveries for N-nitrosomethyl-4-aminobutyric acid (NMBA, 78.0-96.0 %) and 1-methyl-4-nitrosopiperazine (MeNP, 90.0-109.0 %) were just obtained in the opposite condition (with and without formic acid, respectively). In conclusion, we provide a sensitive and reliable method for nitrosamine estimations to ensure the safety of biological medications.