Determination of 5-fluorocytosine, 5-fluorouracil, and 5-fluorouridine in hospital wastewater by liquid chromatography-mass spectrometry.

Chemotherapeutics are pharmaceutical compounds the occurrence of which in the environment is of growing concern because of the increase in treatments against cancer diseases. They can reach the aquatic ecosystems after passing through wastewater treatment plants without complete removal. One of the most frequently used chemotherapeutics is 5-fluorouracil which exhibits a strong cytostatic effect. In this paper, an analytical methodology was developed, validated, and applied to determine 5-fluorouracil, its precursor, 5-fluorocytosine, and its major active metabolite, 5-fluorouridine, in hospital wastewater samples. Due to the expected low concentrations after dilution and interferences present in such a complex matrix, a very selective and sensitive detection method is required. Moreover, an extraction method must be implemented prior to the determination in order to purify the sample extract and preconcentrate the target analytes at micrograms per liter concentration levels. Solid-phase extraction followed by liquid chromatography with tandem mass spectrometry was the combination of choice and all included parameters were studied. Under optimized conditions for wastewater samples analysis, recoveries from 63 to 108% were obtained, while intraday and interday relative standard deviations never exceeded 20 and 25%, respectively. Limits of detection between 61 and 620 ng/L were achieved. Finally, the optimized method was applied to samples from hospital wastewater effluents.

Development of LC-HRMS methods for evaluation of metabolic conversion of 5-fluorocytosine at GDEPT procedure.

Gene-directed enzyme/prodrug therapy represents one of the experimental treatment approaches. The system based on conversion of nontoxic prodrug 5-fluorocytosine to chemotherapeutic 5-fluorouracil by cytosine deaminase or fusion cytosine deaminase::uracil phosphoribosyl transferase belongs to the most frequently used. The detailed analysis of 5-fluorocytosine, 5-fluorouracil and its metabolites enables to understand various responses of tumour cells to treatment as well as mechanisms of resistance. A fast, sensitive and accurate methods based on liquid chromatography with high-resolution mass spectrometry (LC-HRMS) for the identification and quantification of 5-fluorocytosine, 5-fluorouracil and its major metabolites were developed. Two different hybrid high-resolution mass spectrometers sufficient for study of metabolic pathways were used. The LC-ESI IT-TOF MS method was successfully used for identification of 5-fluorocytosine, 5-fluorouracil and its metabolites in complex biological matrices (mesenchymal stromal cells and tumour cells media) and for confirmation of the metabolic conversion of 5-fluorocytosine even in chemoresistant tumour cells media samples. For quantification, the LC-HESI QExactive MS method was developed and validated. The developed method demonstrated a very good linear range for 5-fluorocytosine from 1 ng/mL to 1000 ng/mL and for its major metabolites from 5 ng/mL to 1000 ng/mL. The limits of detection and limits of quantification ranged from 1.1 to 26 ng/mL and from 3.6 to 87 ng/mL, respectively. Both developed methods confirmed the ability of gene-directed enzyme prodrug therapy to metabolically convert 5-fluorocytosine to 5-fluorouracil and its major metabolites in real samples of tumour cell media and mesenchymal stromal cells.