Automated SPE-GC-MS/MS method for the biomonitoring of eight N-nitrosamines in human urine

N-Nitrosamines (NAms) are potent carcinogenic and mutagenic contaminants that may originate from both exogenous exposure and endogenous nitrosation. Human urine is an important non-invasive matrix for biomonitoring NAms exposure; however, the trace concentrations of these compounds and the complexity of the urinary matrix require highly sensitive and selective analytical methods. In this study, a highly automated method based on solid-phase extraction (SPE) coupled with gas chromatography-triple quadrupole tandem mass spectrometry (GC-MS/MS) was developed for the simultaneous determination of eight NAms in urine. By optimizing the SPE sorbent, sample loading strategy, and elution conditions, efficient enrichment and cleanup of the target compounds were achieved using only 4 mL of urine. The automated SPE procedure reduces manual handling, improves sample-preparation reproducibility, and enhances operational efficiency. Method validation showed the recoveries of 80.68% to 111.33%, with intra-day and inter-day precisions (RSDs) below 5.59% and 7.72%, respectively. The limits of detection (LOD) ranged from 0.00335 to 0.01 µg L^-1. The method was applied to 60 urine samples from a community-based population, in which NDMA and NPYR showed the highest detection frequencies. These results indicate that the method is sensitive, repeatable, and analytically stable, and is suitable for biomonitoring trace-level human exposure to NAms.