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 mu g/L with LOQ (limit of quantitation) of 0.5 mu g/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.

Automated summary

Nitrosamine impurities have been detected in pharmaceutical products recently, but no analytical method is available for biopharmaceuticals. In this study, a sensitive and reliable method was developed for quantifying nitrosamine contaminants in antibody drugs, demonstrating accuracy and precision.