A sequence of chromatographic methods (thin-layer chromatography, high-performance liquid chromatography and glass capillary gas chromatography) was used to separate the acid fraction of human urine. The power of this method to separate and detect previously unknown compounds and the elucidation of their final structure with mass spectrometry is exemplified by the identification of N-acetyl-2-aminooctanoic acid as a metabolic compound in the urine of healthy individuals. In addition, the conjugate of glycine with indolepropionic acid, N-formylanthranilic acid, succinoylphenylalanine, delta-hydroxyvaleric acid, beta-hydroxycapric acid, 3-hydroxyadipic acid, and higher homologues were detected in a polar fraction of human urine.