3-(4-bromophenyl)-5-acetyloxymethyl-2,5-dihydrofuran-2-one (LNO-18-22) is a representative member of a novel group of potential antifungal drugs, derived from a natural 3,5-disubstituted butenolide, (-)incrustoporine, as a lead structure. This lipophilic compound is characterized by high in vitro antifungal activity and low acute toxicity. For the purpose of in vivo studies, a new bioanalytical high-performance liquid chromatographic method with UV photodiode-array and mass spectrometric detection (HPLC-PDA-MS), involving a direct injection of diluted mouse urine was developed and used in the evaluation of the metabolic profiling of this drug candidate. The separation of LNO-18-22 and its phase I metabolites was performed in 37 min on a 125 mmx4 mm chromatographic column with Purospher RP-18e using an acetonitrile-water gradient elution. Scan mode of UV detection (195-380 nm) was employed for the identification of the parent compound and its biotransformation products in the biomatrix. Finally, the identity of LNO-18-22 and its metabolites was confirmed using HPLC-MS analyses of the eluate. These experiments demonstrated the power of a comprehensive analytical approach based on the combination of xenobiochemical methods and the results from tandem HPLC-PDA-MS (chromatographic behaviour, UV and MS spectra of native metabolites versus synthetic standards). The chemical structures of five phase I LNO-18-22 metabolites and one phase II metabolite were elucidated in the mouse urine, with two of these metabolites having very unexpected structures.