High-performance liquid chromatography coupled to atmospheric pressure ionization-electrospray ionization mass spectrometry (API-ESI-MS) was investigated for the analysis of corticosterone metabolites; their characterization was obtained by combining the separation on Zorbax Eclipse XDB C-18 column (eluted with a methanol-water-acetic acid gradient) with identification using positive ion mode API-ESI-MS and selected ion analysis. The applicability of this method was verified by monitoring the activity of steroid converting enzymes (20 beta-hydroxysteroid dehydrogenase and 11 beta-hydroxysteroid dehydrogenase) in avian intestines. (C) 1999 Elsevier Science B.V. All rights reserved.