Reversed-phase high-performance liquid chromatographic separation of 14C-labelled toloxatone and its metabolites.

A method for the analytical and micropreparative separation of toloxatone and its urinary metabolites in man is described. Toloxatone was given as an aqueous solution and was labelled with 14C. Following solvent extraction of urine, before and after enzymatic hydrolysis, one-step thin-layer chromatography on silica gel in combination with reversed-phase high-performance liquid chromatography, gave a good micropreparative separation for mass spectrometric analysis. After lyophilization of the high-performance liquid chromatographic fractions, the purity of the metabolites was checked by thin-layer chromatography. Acetic acid was chosen to regulate the pH of the mobile phase (acetonitrile-water) because it can be easily removed by lyophilization when a preparative separation is desired. The retention times as a function of the pH have been evaluated. Formic acid is also proposed for the optimization of the high-performance liquid chromatographic analysis. The quantitative analysis of 14C-labelled toloxatone and its metabolites was carried out, after solvent extraction of 2 ml of urine, using the same high-performance liquid chromatographic method with off-line and flow-through radioactivity detection.