The applicability of in-vial two-phase liquid-phase microextraction (LPME) in porous hollow polypropylene fiber was studied for the sample preparation of unconjugated anabolic steroids in urine. Four different anabolic steroids-metabolites of fluoxymesterone, 4-chlorodehydromethyltestosterone, stanozolol and danazol-were used as test compounds and methyltestosterone as an internal standard. A standard two-phase LPME method for use with liquid chromatography/mass spectrometry (LC/MS) was set up and the influence of different parameters, including the nature of organic solvent, extraction time, salting-out and temperature, on the LPME process was investigated. Taking advantage of the preliminary studies, a novel two-phase LPME method utilizing simultaneous in-fiber silylation was developed and validated for gas chromatographic/mass spectrometric (GUMS) analysis of a danazol metabolite in urine. In all, LPME allowed a very straightforward, simple and selective way to prepare urine samples for steroid analysis, being most suitable for hydrophobic steroids. The LPME method with in-fiber derivatization for GUMS analysis exhibited high sensitivity, repeatability and linearity and enabled simultaneous filtration, extraction, enrichment and derivatization of the analyte from urine matrix without any other steps in sample pretreatment. (c) 2005 Elsevier B.V. All rights reserved.