A method for solid phase extraction and HPLC-MS/MS of the cytostatics 5-fluorouracil, cytarabine, and gemcitabine and human metabolites uracil 1-beta-d-arabinofuranoside and 2',2'-difluorodeoxyuridine in wastewater was established. Wastewater samples from a Swiss hospital were analyzed for 5-fluorouracil, gemcitabine and 2',2'-difluorodeoxyuridine. The limits of quantification were 5.0, 0.9, and 9.0ng/L and the maximum concentrations detected were 27, 38, and 840ng/L, respectively. Along with the method development, retention mechanisms on the hydrophilic interaction chromatography (HILIC) stationary phase were studied. Both partitioning and adsorption play a role in the retention on the tested sulfoalkylbetaine modified silica HILIC column material. The contribution of these two processes is changing over the 1.6-40% range water in the mobile phase. Although the specific break point is difficult to determine, adsorption becomes more significant as the fraction of water in the mobile phase decreases below approximately 16%.