Simultaneous determination of areca nut- and tobacco-specific alkaloids in saliva by LC-MS/MS: Distribution and transformation of alkaloids in oral cavity

Areca nut and tobacco are frequently used in combination. Cigarette smoking and betel quid (BQ) chewing habits impose greater oral cancer risk than either habit alone. Saliva is a better noninvasive diagnostic material as it is in direct contact with oral mucosa and cancerous lesions. This study describes the application of isotope-dilution LC-MS/MS for simultaneous quantitation of five areca nut-specific alkaloids (ASAs) and three tobacco-specific alkaloids (TSAs) in human saliva. With this method, we demonstrate that the distribution of ASAs vary significantly in smokers who chew BQ habitually, due to the hydrolysis of ASAs and metabolic activity in the oral cavity. The alkaline condition formed due to slaked lime in BQ, plays an important role in the distribution of ASAs and TSAs, by catalyzing the hydrolysis of ester forms of ASAs to their respective carboxylic acid forms besides facilitating the TSA (i.e., nicotine) absorption in the oral cavity. Moreover, our results reveal that oral mucosa rather than saliva contributes to the metabolism of ASAs at oral cavity. Less than 2.1% of ASAs were metabolized by saliva, as determined by in vitro test. Our findings may provide a better insight into the pathobiology of oral carcinogenesis due to BQ chewing.