Smokeless tobacco (ST) products are used worldwide, and consumption is increasing in the USA. Although ST products are considered to occupy a different position on the tobacco product continuum of risk compared to combusted tobacco products, they can still lead to health problems, including cancer, dental problems and changes in heart rate and blood pressure. Therefore, the determination of harmful and potentially harmful constituents released from ST products into human saliva is important. Four certified reference ST products were tested in this study: loose leaf chewing tobacco (3S1), Swedish-style snus (1S4), snus (1S5) and moist snuff (3S3). These certified reference ST products are manufactured for research purposes, not for human consumption. The reference ST products were used in this study because they have been well characterized and are intended and designed to represent commercial ST products. The reference ST products were incubated in human saliva at 37 degrees C with a range of incubation times for the evaluation of constituents released from these products into human saliva. In this study, alkaloids (nicotine and cotinine), tobacco-specific N-nitrosamines (TSNAs) (N'-nitrosornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone) and benzo[alpha]pyrene (B[alpha]P) in the reference ST products and saliva samples were determined by gas chromatography-mass spectrometry (GC-MS), gas chromatography-fame ionization detection (GC-FID), or ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS-MS). Our results indicate that the amounts of each constituent released from the reference ST products were altered by the tobacco cut size and product format (pouched or unpouched). The constituents (TSNAs and alkaloids) in moist snuff and loose leaf chewing tobacco were released faster compared to those in Swedish-style snus and snus. B[alpha]P was only detected in reference moist snuff samples, and only 3.4% of the total B[alpha]P was released into human saliva after incubation for 60 min, whereas higher percentages of total TSNAs and alkaloids were released at different rates from the four reference ST products.
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