High performance liquid chromatographic determination of N-nitrosoamines by pre-column fluorescence derivatization with acridone- N-acetyl chloride

A sensitive high performance liquid chromatographic method for the determination of N-nitrosoamines with pre-column fluorescence derivatization has been developed. N-nitrosoamines are first changed into secondary amines using denitrosation reagent, then react with acridone- N-acetyl chloride (ARC-Cl) to produce corresponding secondary amine derivatives, which exhibit a strong fluorescence. Maximum emission for ARC derivatives is 430 nm ( λ ex 404 nm). The labelled derivatives are very stable, less than 4% decomposition occurs after heating at 40°C for 24 h. Fluorescence intensities of derivatives are higher in neutral and alkaline than in acidic solutions. This method, in conjunction with a multi-gradient program, offers a baseline resolution of the ARC derivatives from a linear acetonitrile gradient. Separation is carried out on a reverse phase C 18 column. Derivatization and chromatographic conditions are optimized. The relative standard deviation ( n=6) at an analytical concentration of 10 pmol of each N-nitroamine is less than 4.5%. The detection limits at the fmol level. The method described is also suitable for analysis of other amino compounds in different biological samples.