Nationwide investigation on the use of new psychoactive substances in Italy through urban wastewater analysis.

New psychoactive substances (NPS) emerged in the mid-2000s as a legal alternative to established illicit drugs. Despite the high individual and public harm associated to NPS, little is known about their real extent of use. New strategies are required to deal with the challenging monitoring of NPS, affected by the high number of substances available in the market, their rapid change and level of innovation, and their easy distribution mainly through the web. In this study, a wastewater-based epidemiology (WBE) approach was applied for a nationwide monitoring of the use of eight categories of NPS in the population, including fentanyl analogues. Sixty-two biomarkers of NPS were selected following an established criterion, that included the most frequently and recently reported. A selective analytical method based on solid-phase extraction and liquid chromatography-tandem mass spectrometry was developed and validated for NPS analysis in wastewater. Composite wastewater samples (24 h) were collected in 33 Italian cities in October-November 2020 and analyzed according the validated method. Results highlighted the use of ten NPS, mainly synthetic cathinones and tryptamines, all over Italy. Methcathinone was found in all the cities and the highest mass loads corresponded to 3-methylmethcathinone with values up to 3.8 mg/day/1000 inhabitants. Low levels of fentanyl (found in 9 cities) and its main metabolite norfentanyl (11) were found whereas no fentanyl analogues were identified. As far as we know, this is the first time that the use of fentanyl and its analogues was investigated in Italy by wastewater analysis. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection.