LC-HRMS profiling of processing-induced formation of carcinogenic N-nitrosamines in Areca nut products

Areca nut (AN) chewing is a major risk factor for oral cancer, partly due to carcinogenic areca nut-specific N-nitrosamines (ASNAs). While ASNAs are thought to form via nitrosation of areca nut-specific alkaloids (ASAs) by oral nitrite, their inherent presence in AN products remains unclear. We developed and validated an LC-HRMS method to quantify five ASNAs simultaneously. Thirty-eight AN samples, including raw materials (RMs) and commercial products (CPs) from India, China, and Taiwan, were analyzed. At least one ASNA was detected in all samples (0.39-242 pmol/g), with N-nitrosoguvacoline predominant, followed by N-nitrosoguvacine, 3-(N-nitrosomethylamino)propionitrile, and N-nitrosonipecotic acid; 3-(N-nitrosomethylamino)propanal was not detected. CPs contained higher ASNA levels than RMs, showing up to 133-fold and 272-fold increases in Gutkha and Yanguo, respectively. ASNA levels correlated with ASAs and nitrite (p < 0.001). These findings suggest that processing or additives enhance ASNA formation through nitrosating agents, emphasizing the need for monitoring to reduce potential risks.