Field trials were conducted in 2016, 2017 and 2018 with dark fire-cured, dark air-cured, and burley tobacco at Princeton, Murray and Lexington Kentucky. Tobacco variety used in 2016 was a low converter (LC) variety, varieties used in 2017 and 2018 were LC and higher converter (HC) varieties. Potassium sources used at all locations and in all years were potassium sulfate (K2SO4) and potassium chloride (KCl). Application rates used at all locations and in all years were 93, 186, 279 kg K ha-1along with an untreated control that received no potassium. In all trials, tobacco that was treated with either potassium source yielded numerically higher than the untreated control. In seven out of 10 trials, LC varieties had a higher moisture content than HC varieties. Tobacco treated with KCl had higher chloride levels than tobacco treated with K2­SO4.Quality grade index was similar for tobacco treated with KCl compared to tobacco treated with K2SO4. In all trials, tobacco treated with KCl had numerically lower Tobacco Specific Nitrosamines (TSNA) levels than tobacco treated with K2SO4. Reductions in TSNA levels were 30% lower in tobacco treated with KCl compared to tobacco treated with K2SO­4.

Tobacco-specific nitrosamines (TSNA) are carcinogenic constituents derived from alkaloids in tobacco. Researchers are actively exploring several avenues to reduce TSNA levels in tobacco products like moist snuff tobacco. The focus of the research presented within is the quantitative analysis of TSNA in tobacco, specifically N’-nitrosonornicotine (NNN), 4-(methylnitrosamino)-1(3-pyridyl)-1-butanone (NNK), N’-nitrosoanatabine (NAT), and N’-nitrosoanabasine (NAB). Tobacco alkaloids and nitrosamines in tobacco are currently analyzed by different instrumentation due to orders of magnitude difference in their concentrations, chromatographic separation challenges due to structural similarities, and similar mass fragmentation patterns. An analytical column using silica and 1,2-bis(siloxy)ethane hybrid particles of 1.7 µm size is the foundation of a chromatographic separation of NNN, NNK, NAT, NAB, nicotine, nornicotine, anatabine, and anabasine. This is the first rapid and robust quantitative method for the TSNA and their alkaloid precursors using high pH mobile phase conditions with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). The suitability of the method is demonstrated by its application to the analysis of reference tobacco materials for cigarettes and moist snuff. In addition, a novel TSNA analytical method was developed using TSNA-specific molecularly imprinted polymers (MIP) as the selective extraction element from tobacco extract. The affinity mechanisms between MIP and TSNA were found to have extensive cross-reactivity to structurally similar alkaloids present in tobacco extract. TSNA-specific MIP was demonstrated to have stronger retention for the alkaloids than for the TSNA substrate. The MIP-TSNA interaction was optimized to create the first analytical method to quantify underivatized NNN and NNK from tobacco extracts by HPLC-UV.

Significant variability in cured leaf tobacco-specific nitrosamine (TSNA) content is commonly observed when sampling within dark air-curing barns. This variability may be due to inconsistency in the curing environment within different areas of the barn. A study was initiated in 2012 through support from a CORESTA Study Grant to evaluate if leaf TSNA content is related to microenvironmental conditions in the barn. Seed screened for low conversion of nicotine to nornicotine (sc) and high converter (HC) selections of TR Madole dark tobacco were cured in barns near Princeton and Lexington, Kentucky in 2012 and 2013. Temperature and relative humidity were measured with data loggers placed at 27 locations within each barn for the duration of curing. TSNA content was determined from 20-leaf samples collected from each selection at each of the 27 locations within each barn. There were no significant effects of individual data logger placement in either variety selection on hours above 24°C temperature, hours above 80% relative humidity, or TSNA; therefore, we investigated these data within 3-dimensional aspects of tier, room, and bent within each barn. There were various effects of tier, room, and bent on temperature, relative humidity, and TSNA; but limited significant relationships between temperature, relative humidity, and TSNA.