4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the most carcinogenic tobacco-specific nitrosamines (TSNAs) and one of the more important compounds in FDA’s HPHCs list. It is formed from PON, an oxidation product of nicotine. Accurate measurement of PON is essential to understand the formation pathway of NNK and further reduce it in tobacco. In the literature, there is little information on analysis of PON in tobacco leaf and tobacco products. A LC/MS/MS method was developed to determine PON in tobacco and tobacco products.

Tobacco was extracted by citric acid and disodium phosphate buffer, pH 3.0, for 45 min with shaking. The extract solution was filtered through a 0.22 µm PTFE filter to remove the tobacco powder and injected into Waters Xevo TQD LC/MS/MS. If necessary, the extract solution can be cleaned-up by methylene chloride. All analysis was done on a Waters ACQUITY UPLC BEH C18 2.1 x 150 mm column with 1.7 µm particles. Separation was achieved using a gradient mobile phase consisting of 10mM ammonium acetate with 0.1% NH₄OH and acetonitrile.

Preliminary experimental data demonstrated that LC/MS/MS can be used for quantitative analysis of PON in tobacco and tobacco products with high selectivity, sensitivity and speed. Good linear response, R2 = 0.999, was measured within the linear dynamic range of 20 - 2000 ng/ml PON. The recovery of PON was ~105%. Total analysis time for each sample was 8 min. The coefficients of variation for tobacco samples are under 10%. The limits of detection (LOD) and limit of quantification (LOQ) were 7 and 20 ng/ml, respectively. This method has been applied to tobacco leave and cigarette filler.