Evaluation of human hepatocyte incubation as a new tool for metabolism study of androstenedione and norandrostenedione in a doping control perspective

A novel and simple method has been developed for the simultaneous quantification of tryptophan, kynurenine and indole derivatives as well as four catecholamines, including dopamine, noradrenaline, homovanillic acid and 3,4-dihydroxyphenylacetic acid. The method utilises isocratic reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase and the detection potentials, was investigated. Separation of 13 compounds was achieved by a mobile phase consisting of 10% methanol in 50 mM sodium phosphate-acetate buffer, pH 4.10, containing 0.42 mM octanesulphonic acid. The calibration curve was linear over the range 12 pg to 300 ng on-column. The detection limits (SIN 3) depended on the working potential and were found to be between 10 and 100 pg injected. The method was reproducible with intra-day RSDs of 0.3 to 1.5% and inter-day RSDs of 0.5 to 4%.