Separation of N-nitrosoamino acids by C18 reversed-phase ion-pair high-performance liquid chromatography and compatible detection by electrospray ionization mass spectrometry.

Four non-volatile N-nitrosoamino acids, namely N-nitrososarcosine, N-nitrosoproline, N-nitrosothiazolidine-4-carboxylic acid and N-nitroso-2-methyl-thiazolidine-4-carboxylic acid were separated by C18 reversed-phase ion-pair high-performance liquid chromatography (HPLC) using 1.4 mM C16-cetyltrimethylammonium chloride in methanol-water-acetonitrile (60:35:5, v/v) as the mobile phase. The N-nitrosoamino acids were sensitively detected by negative electrospray ionization mass spectrometry (ESI-MS) in the form of the deprotonated carboxylate anion, [M-H]-. Compatibility problems associated with HPLC separation and ESI-MS detection, such as formation of solvent cluster ions and the effects of co-eluting anions of the ion-pairing reagent, were systematically investigated. The optimized experimental conditions for separation and detection of N-nitrosoamino acids were described.