SENSITIVE ASSAY SYSTEM FOR NITROSAMINES UTILIZING HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH PEROXYOXALATE CHEMILUMINESCENCE DETECTION

A high-performance liquid chromatographic system in combination with postcolumn chemiluminescence detection for the determination of nitrosamines was developed. The stability of the chemiluminogenic reagent solution was investigated using a conventional high-performance liquid chromatograph with a UV-Vis spectrophotometric detector, and the optimum conditions for chemiluminescence intensity were also established with a single reagent pump postcolumn detector. The sample was first denitrosated with hydrobromic acid-acetic acid to produce the corresponding secondary amines, which were then subjected to reaction with dansyl chloride to form dansyl derivatives. The reaction mixtures were separated on a Perkin-Elmer HS3 C18 reversed-phase column with acetonitrile-water (63.5:36.5, v/v) as mobile phase, with 3.0 mmol/l of imidazole added as a catalyst for chemiluminescence and with the pH adjusted to 6.2 with oxalic acid to give a better separation, followed by detection with a postcolumn chemiluminescence detector using bis(2-nitrophenyl) oxalate and hydrogen peroxide as chemiluminogenic reagents. The sensitivity of this method was more than 120 times greater than that of fluorescence detection and four orders of magnitude greater than that of UV-Vis spectrophotometric detection. The detection limits with this procedure at a signal-to-noise ratio of 4 were between 0.31 and 1.20 pg and the relative standard deviations were between 2.8% and 6.7% for six nitrosamines. The linearity of the calibration graphs and the correlation coefficients were good.