High-performance liquid chromatographic analysis of metabolites of the nicotine-derived nitrosamines, N′-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone

An improved high-performance liquid chromatographic system was developed for separation of 11 metabolites of the nicotine-derived nitrosamines N′-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). The new system employed a 5-μm octadecylsilane bonded column eluted with aqueous sodium acetate-methanol gradients of varying pH. Analysis times were typically 30 min for NNN metabolites and 50 min for NNK metabolites, compared to 80 and 90 min, respectively, when 10-μm columns were used. The E and N isomers of all nitrosamine-containing metabolites of NNK were separated. The chromatographic behavior of the 11 metabolites as well as NNN and NNK was studied between pH 4.0 and 7.5. The retention times of several metabolites were altered significantly as a function of pH. The results of the pH study provide valuable additional criteria for metabolite identification as well as optimized conditions for their separation. Applications of the system to the metabolism of [2′-¹⁴C]NNN in cultured rat esophagus and [carbonyl-¹⁴C]NNK in rat liver slices are presented.