A comprehensive approach for N-nitrosamine determination in pharmaceuticals using a novel HILIC-based solid phase...

N-nitrosamines (NAs) are potentially highly carcinogenic compounds that have recently been detected in traces in various drug products (DPs). Due to the different physicochemical properties of NAs and active pharmaceutical ingredients (APIs), there is a lack of appropriate analytical methods for simultaneously determining multiple NAs in various DPs. To overcome these limitations, a versatile and innovative analytical approach was developed using a unique sample clean-up procedure by solid phase extraction based on hydrophilic interaction chromatography, which retains high amounts of APIs and polar excipients while allowing NAs of interest to pass through. The samples were analyzed by liquid chromatography coupled with electrospray ionization high-resolution mass spectrometry. The proposed highly sensitive, selective, and robust method was successfully validated, resulting in excellent linearity (R² > 0.999), accuracy (85-115 %), and precision (RSD <10 %) with adequate recoveries (>80 %), achieving limits of quantitation of at least 42.5 % of regulatory limits. Furthermore, robustness was confirmed for ten DPs (recoveries >80 % and RSD <15 % for all NAs), including those containing up to three APIs. The analytical approach was utilized to examine 26 commercially available and expired DPs. Three NAs (N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine, and N-nitroso-di-n-butylamine) were detected, only NDMA exceeded the limits in expired DPs by up to 32-fold. It was found that special care should be taken when handling samples as NDMA content can be decreased by almost 50 % if samples are not prepared immediately. The approach was tested on 59 different APIs and was confirmed as reliable tool for routine monitoring of 15 NAs in various DPs. Due to its flexibility, the method can be further adapted to the specific API of interest or extended to the newly emerging NA drug substance-related impurities to ensure the safety of DPs and thereby mitigate potential health risks.