Dottorato in SCIENZE FARMACEUTICHE

Titolo della tesi: High Resolution Mass Spectrometr: new methods of analysis for risk assessment by cyanotoxins and cyromazine in water for human consumption

The recent implementation of the EU Directive 1787/2015 introduced in Italy the Water Safety Plan (WSP), a preventive model to guarantee drinking water through control measures extended to the entire water supply chain, from collection, treatment and distribution to the user. In this context of prevention, protection and monitoring of the quality of water intended for human consumption, it becomes important to develop analytical methods that allow the search for an increasing number of contaminants that could be present in the water, with particular attention to substances considered emerging contaminants (EPs). Emong the EPs, a wide range of natural and man-made chemicals that currently are not included in routine monitoring programmes but that could have nocive effects on human health, my attention has focused on the secondary metabolites of cyanobacteria and on Cyromazine. The proliferation of cyanobacteria in water used for human consumption is an emerging issue in Italy in recent years, because they can produce undesirable or toxic metabolites (cyanotoxins), affecting the quality of water and causing a significant health risk. Cyromazine, instead, is a growth regulating insecticide but the recent issue of a press released by the Directorate General for Hygiene and Safety of Food and Nutrition revokes the authorizations of plant protection products containing it, following the expiry of its Community approval period pursuant to Regulation (EU) 844/2012. In the first part of the research work of this PhD, an analytical method of solid phase extraction (SPE) and subsequent determination in ultra-high performance liquid chromatography (UPLC) coupled to a high resolution mass spectrometer (QTOF) for the simultaneous determination of 21 cyanotoxins in water for human consumption was developed. In the second part of the research the developed method was validated, resulting robust in terms of repeatability, reproducibility and linearity, with recovery percentages above 85% and with relative standard deviations ≤16% and with LODs in the range 0.002 to 0.047 ug/l. This method allows the simultaneous identification of 21 target compounds, in a short time of analysis (16 minutes) and low injection volume (10 µl); it was then applied during a monitoring activity in an Italian volcanic lake in Viterbo (Lazio Region, Italy), due to a severe algal proliferation in January 2018 – January 2020 period and the obtained results were used for the assessment of cyanobacteria proliferation risk and of cyanotoxin production in drinking water chain. The method results to be fully functional to the more advanced preventive approach to control cyanotoxins within the drinking water supply chain according to the Water Safety Plan (WSP) model, as it is applied to raw and tap water, by allowing an early detection of simultaneous algal toxin production, the actual effectiveness of mitigation measures and the adoption of the UPLC-HRMS approach as validation/confirmatory approach for on-line monitoring of algal and toxins in water bodies and water distribution systems. Moreover the development of another analytical method without SPE for the determination of further two cyanotoxins (Anatoxin-a and Cylindrospermpsin) has been started but the method is not complete and needs a further optimization step. At last the research activity was focused on the development of a method in UPLC/HMRS for the identification and quantification of ciromazyne in drinking water samples. The method was developed and validated, resulting robust, precise and accurate with a relative standard deviation ≤10%, a good linearity in the range 2.5 - 50 μg/l and LOD equal to 0.5 ug/l.Since the use of pesticides containing cyromazine will be allowed until June 2021, the developed method result to be useful to verify the absence of this compound in water.