Not only eliminated pharmaceuticals, but also their metabolites may pose a risk to the environment because of their preserved pharmacological activity which may adversely affect the human health and other exposed organisms. Therefore, the aim of this Master's thesis was to develop a rapid and highly sensitive method for quantification of drugs' metabolites frequently found in Slovenian wastewaters.
Wastewater samples were prepared with solid-phase extraction method using a semi-automated system SPE-DEX and analysed by LC-MS/MS. Analyte concentrations were calculated using the standard addition method, which also eliminated the influence of matrix effect on the accuracy of quantification. The method was successfully optimised and validated in terms of accuracy, precision, working range, limit of quantification, recovery and matrix effect. We confirmed its reliability and high sensitivity for the simultaneous analysis of 26 analytes (11 pharmaceuticals from multiple therapeutic groups and 15 of their metabolites), which were selected based on a literature review and preliminary analyses. The method was applied to twelve real wastewater samples (three influent and nine effluent) that were collected from nine municipal wastewater treatment plants from several Slovenian regions. Out of the total 26 analytes, 12 were detected in all samples. Considering only metabolites, 60 % were detected in all samples and 80 % in at least one sample. The highest average concentrations in influent and effluent samples were measured for caffeine (4.1 and 4.6 µg/L), 1,7-dimethylxanthine (3.6 and 2.7 µg/L) and salicylic acid (1.1 and 1.7 µg/L), respectively. Carbamazepine was measured in all samples in average concentrations of 165.9 ng/L in influent and 142.2 ng/L in effluent samples, respectively. Diazepam, fluoxetine, norfluoxetine, paliperidone and endoxifen were not detected in any sample. In general, the measured concentrations were lower or comparable to those reported in similar studies worldwide. For the wastewater treatment plants from which both influent and effluent samples were collected, the removal efficiencies of the analytes were evaluated. Concentrations in effluents decreased compared to influents, except for diclofenac and carbamazepine, which were found to have low or even negative removal efficiencies.
In conclusion, the measured concentrations in effluents presumably do not pose a direct risk to humans. However, the metabolites, especially those with preserved pharmacological activity, should be included in future ecotoxicological studies due to their impact on aquatic organisms.