Spe-Uhplc-Esi-Ms/Ms Method Development For 5-Fluorocytosine, 5-Fluorouracil And 5-Fluorouridine Analysis In Complex Environmental Samples

Abstract

5-fluorocytosine (5FC) is an anti-mycoticum, which effect is based on metabolic conversion to 5-fluorouracil (5FU). 5FU is a compound with cytostatic effect based on transformation to other metabolites like 5-fluorouridine (5FURD). Also, metabolic conversion of 5FC to 5FU is used in cancer treatment procedure called "suicide gene therapy" [1]. In recent years, the use of ultra-high performance liquid chromatography (UHPLC) coupled with mass spectrometry (MS) is dominated for purpose of identification and determination of 5FC, 5FU and its metabolites in complex environmental samples [2,3]. All hospital wastewater samples were analyzed by UHPLC-QqQ mass spectrometer (Waters) with electrospray ionization. The electrospray worked in positive and negative ionization mode with capillary voltage ±3 kV and ion source temperature was set to 150 ˚C. Chromatographic separation was performed on Luna Omega Polar C18 (50x2.1 mm; 1.6 μm) column (Phenomex). Due complexity of wastewater samples, it was necessary to use solid phase extraction (SPE). All SPE extractions were performed on Isolute ENV+ (500 mg / 6 mL) cartridges (Biotage).Parameters like sample volume, pH and increasing ionic strength were studied. Sample volume was tested in range 50 - 250 mL and 75 mL was selected as optimal. The influence of pH on separation was performed in range 2 – 8. The best results were obtained at pH 4.5. The increasing of ionic strength was led to worse retention of target analytes. For improvement of analytes ionization, different additives to mobile phase were tested and final composition of mobile phase was 0.1% AcA : MeOH. Basic analytical parameters of the developed method like recovery, inter-day and intraday repeatability on model mixtures and for consideration of matrix effect in wastewater were tested. The developed SPE-UHPLC-ESI-MS/MS method was applied for analysis of wastewater samples from hospital oncology department and from whole hospital.