Determination of the urinary concentrations of six bisphenols in public servants by online solid-phase extraction-liquid chromatography tandem mass spectrometry.

Bisphenols are widely used as monomers and additives in plastic production. Thus, bisphenol A (BPA) and its most prominent substitutes have been detected in many environmental and human samples. This study proposes an online solid-phase extraction analytical methodology coupled to liquid chromatography with tandem mass spectrometry for the determination of six bisphenols (BPA and bisphenols F (BPF), S (BPS), AF (BPAF), B (BPB), and E (BPE)) in urine samples as an efficient and automated methodology. The method was developed and validated for all bisphenols with good recoveries (92-112%) and repeatability (RSD ≤ 10%) despite the variable matrix effects, except BPAF (which would require a dedicated internal standard), achieving method quantification limits in the 0.05-2.2 ng mL-1 range. The methodology was subsequently applied to 435 urine samples from a non-occupational exposure population (civil servants for the regional government) from Santiago de Compostela (Galicia, Spain). Only BPA, BPF, and BPS were positively detected; the last two presented higher detection frequencies than BPA. When the urinary concentrations are extrapolated to human intake and compared to the European Food Safety Agency (EFSA) tolerable daily intake (TDI) of 2 × 10-4 µg kg-1 day-1 (TDI), all BPA positively identified samples would surpass this threshold. Although no TDI exists currently for the other two identified bisphenols, it is evident that human exposure to bisphenols should be limited. Finally, the results stratification by gender revealed higher levels of exposure to BPF in the women group.

Wastewater-based epidemiology methodology to investigate human exposure to bisphenol A, bisphenol F and bisphenol S.

Wastewater-based epidemiology (WBE) has become an invaluable tool for tracking the evolution of use or exposure of/to numerous substances. Bisphenols, commonly utilized in manufacturing plastic goods, have been categorized as endocrine disrupting chemicals, underscoring the critical need for real-time data on their local-level exposure to safeguard public health. In this study, we have developed a novel analytical method and WBE framework for the assessment of population-level exposure to bisphenol A (BPA) and its most prominent substitutes, bisphenols F and S (BPF and BPS), through the determination their Phase II metabolites in wastewater by WBE. Stability and exclusivity tests denoted that glucuronides are not stable in sewage, whereas sulfate metabolites are good biomarkers. Therefore, a solid-phase extraction followed by liquid chromatography-tandem mass spectrometry method was developed for the bisphenols' monosulfates and BPA bissulfate. The analytical method was validated with three different wastewater matrices, providing trueness (as recovery) in the 79-112 % range with relative standard deviations < 12 %, and method quantification limits below 2 ng L-1 for monosulfates, but higher (35 ng L-1) for BPA bissulfate. Subsequently, the method was applied to 24h-composite raw wastewater samples collected over a week in 4 different locations in Spain and Portugal. BPA bissulfate was not detected, but the three monosulfate metabolites of each bisphenol were positively detected in the samples, being the metabolite of BPA the most prevalent, followed by those of BPF and BPS. Community-wide BPA intake was then estimated to be higher than the European Food Safety Agency (EFSA) tolerable daily intake (TDI) of 2 × 10-4 µg kg-1day-1 in all locations. In the case of BPF and BPS, there is not enough metabolism data or even established limit, but they would also surpass safe levels in several locations if a similar metabolism and TDI would be assumed. This innovative method could be used to a larger set of wastewater-treatment plants as an early-warning approach on human exposure to bisphenols.

In vitro biotransformation of 3-methylmethcathinone (3-MMC) through incubation with human liver microsomes and cytosol and application to in vivo samples.

Synthetic cathinones are the second largest group of new psychoactive substances (NPS) monitored by the European Monitoring Centre for Drugs and Drug Addiction. Although 3-methylmethcathinone (3-MMC, C11H15NO) is legally banned in many countries, it is readily available for purchase online and on the street. Due to the scarcity of information regarding the pharmacokinetic and toxicological profile of 3-MMC, understanding its biotransformation pathways is crucial in determining its potential toxicity in humans and in the development of analytical methods for screening of human matrices. To gain more insight, Phase I and Phase II in vitro biotransformation of 3-MMC was investigated using human liver microsomes and human liver cytosol. Suspect and non-target screening approaches were employed to identify metabolites. To confirm in vitro results in an in vivo setting, human matrices (i.e., plasma, urine, saliva and hair) positive for 3-MMC (n=31) were screened. In total three biotransformation products were identified in vitro: C11H15NO2 (a hydroxylated derivate), C11H17NO (a keto-reduced derivate) and C10H13NO (an N-desmethyl derivate). All three were confirmed as human metabolites in respectively 16 %, 52 % and 42 % of the analysed human samples. In total, 61 % of the analysed samples were positive for at least one of the three metabolites. Interestingly, three urine samples were positive for all three metabolites. The presence of 3-MMC in saliva and hair indicates its potential applicability in specific settings, e.g., roadside testing or chronic consumption analysis. To our knowledge, C11H17NO was not detected before in vivo. Although some of these metabolites have been previously suggested in vitro or in a single post mortem case report, a wide in vivo confirmation including the screening of four different human matrices was performed for the first time. These metabolites could serve as potential human biomarkers to monitor human 3-MMC consumption effectively.

Investigation of in vitro biotransformation of tris (1-chloro-2-propyl) phosphate and confirmation in human urine.

Tris (1-chloro-2-propyl) phosphate (TCIPP) is one of the major organophosphate flame retardants present in the indoor and outdoor environment. Knowledge of biotransformation pathways is important to elucidate potential bioavailability and toxicity of TCIPP and to identify relevant biomarkers. This study aimed to identify TCIPP metabolites through in vitro human metabolism assays and finally to confirm these findings in urine samples from an occupationally exposed population to propose new biomarkers to accurately monitor exposure to TCIPP. TCIPP was incubated with human liver microsomes and human liver cytosol to identify Phase I and Phase II metabolites, by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Using a suspect-screening approach, the established biomarkers bis (1-chloro-2-propyl) hydrogen phosphate (BCIPP) and 1-hydroxy-2-propyl bis (1-chloro-2-propyl) phosphate (BCIPHIPP) were identified. In addition, carboxyethyl bis (1-chloro-2-propyl) phosphate (TCIPP-M1), bis (1-chloropropan-2-yl) (-oxopropan-2-yl) phosphate (TCIPP-M2) and 1-chloro-3-hydroxypropan-2-yl bis (1-chloropropan-2-yl) phosphate (TCIPP-M3) were identified. TCIPP-M2, an intermediate product, was not reported before in literature. In urine samples, apart from BCIPP and BCIPHIPP, TCIPP-M1 and TCIPP-M3 were identified for the first time. Interestingly, BCIPP showed the lowest detection frequency, likely due to the poor sensitivity for this compound. Therefore, TCIPP-M1 and TCIPP-M3 could serve as potential additional biomarkers to more efficiently monitor TCIPP exposure in humans.

Use of illicit drugs, alcohol and tobacco in Spain and Portugal during the COVID-19 crisis in 2020 as measured by wastewater-based epidemiology.

The COVID-19 pandemic spread rapidly worldwide in the year 2020, which was initially restrained by drastic mobility restrictions. In this work, we investigated the use of illicit drugs (amphetamine, methamphetamine, ecstasy, cocaine and cannabis), and licit substances of abuse (alcohol and tobacco) during the earlier months (March-July 2020) of the pandemic restrictions in four Spanish (Bilbao and its metropolitan area, Vitoria-Gasteiz, Castellón and Santiago de Compostela) and two Portuguese (Porto and Vila do Conde) locations by wastewater-based epidemiology (WBE). The results show that no methamphetamine was detected in any of the locations monitored, while amphetamine use was only detectable in the two locations from the Basque Country (Bilbao and its metropolitan area and Vitoria-Gasteiz), with high estimated average usage rates (700-930 mg day-1 1000 inhabitant-1). The remaining substances were detected in all the investigated catchment areas. In general, no remarkable changes were found in population normalized loads compared to former years, except for cocaine (i.e. its main metabolite, benzoylecgonine). For this drug, a notable decrease in use was discernible in Castellón, while its usage in Porto and Santiago de Compostela seemed to continue in a rising trend, already initiated in former years. Furthermore, two events of ecstasy (3,4-methylenedioxymethamphetamine, MDMA) dumping in the sewage network were confirmed by enantiomeric analysis, one in Santiago de Compostela just prior the lockdown and the second one in the Bilbao and its metropolitan area in July after relieving the more stringent measures. The latter could also be associated with a police intervention. The comparison of WBE with (web) survey data, which do not provide information at a local level, points towards contradictory conclusions for some of the substances, thereby highlighting the need for stable WBE networks capable of near real-time monitoring drug use.

Comprehensive determination of phthalate, terephthalate and di-iso-nonyl cyclohexane-1,2-dicarboxylate metabolites in wastewater by solid-phase extraction and ultra(high)-performance liquid chromatography-tandem mass spectrometry.

Plasticizers are chemical compounds used in the production of flexible plastics for a large variety of applications. They are present in most of the environments and, hence, we are highly exposed to them via several routes (ingestion, inhalation, etc). Due to the endocrine disruption potential of some of these chemicals and the unknown toxicological effects of their alternatives, assessing human exposure to these contaminants is an issue of emerging concern. Herein we propose an analytical methodology for the determination of several plasticizer metabolites in wastewater as a non-invasive, cheap, and fast exposure monitoring tool complementary to the analysis of urine. A solid-phase extraction procedure followed by an ultra(high)-performance liquid chromatography-tandem mass spectrometry method was optimized and validated for 21 analytes among phthalate, terephthalate, and di-iso-nonyl cyclohexane-1,2-dicarboxylate metabolites. Method quantification limits ranged from 0.079 to 4.4 ng L-1. The method was applied to the analysis of seven daily composite wastewater samples collected in the NW of Spain. Metabolites of low molecular weight phthalates and of di-2-ethylhexyl phthalate were quantified in all samples, despite the existing regulations limiting the use of phthalates. Metabolites of terephthalates, introduced at the end of the 20th century as phthalate substituents, were also quantified in all samples, being the first time that they were detected in this matrix. Exposure back-calculation highlighted di-2-ethylhexyl terephthalate as the second most common plastic additive after diethyl phthalate in the population considered, reflecting the increasing substitution of di-2-ethylhexyl phthalate by its analogous terephthalate.

Source identification of amphetamine-like stimulants in Spanish wastewater through enantiomeric profiling.

Amphetamine (AMP), methamphetamine (MAMP) and 3,4-methylenedioxymethamphetamine (MDMA) occur in wastewater not only as a result of illicit consumption, but also, in some cases, from prescription drug use or by direct drug disposal into the sewage system. Enantiomeric profiling of these chiral drugs could give more insight into the origin of their occurrence. In this manuscript, a new analytical methodology for the enantiomeric analysis of amphetamine-like substances in wastewater has been developed. The method consists of a solid-phase extraction (SPE) followed by liquid chromatography-triple quadrupole-tandem mass spectrometry (LC-MS/MS), which showed low quantification limits in the 2.4-5.5 ng L-1 range. The LC-MS/MS method was first applied to characterize a total of 38 solid street drug samples anonymously provided by consumers. The results of these analysis showed that AMP and MDMA trafficked into Spain are synthesized as racemate, while MAMP is exclusively produced as the S(+)-enantiomer. Then, the analytical method was employed to analyse urban wastewater samples collected from the wastewater treatment plants (WWTPs) of five different cities in 2018 and 2019. Consumption estimated through normalized population loads in wastewater showed an increased pattern of AMP use in the Basque Country. Furthermore, the enantiomeric profiling of wastewater samples was contrasted to lisdexamfetamine (LIS) and selegiline (SEL) prescription figures, two pharmaceuticals which metabolize to S(+)-AMP, and to R(-)-AMP and R(-)-MAMP, respectively. From this analysis, and considering uncertainties derived from metabolism and adherence to treatment, it was concluded that LIS is a relevant source of AMP in those cases with low wastewater loads, i.e. up to a maximum of 60% of AMP detected in wastewater in some samples could originate from LIS prescription, while SEL does not represent a significant source of AMP nor MAMP. Finally, removal efficiencies could be evaluated for the WWTP (serving ca. 860,000 inhabitants) with higher AMP influent concentrations. The removal of AMP was satisfactory with rates higher than 99%, whereas MDMA showed an average removal of approximately 60%, accompanied by an enrichment of R(-)-MDMA.

The embodiment of wastewater data for the estimation of illicit drug consumption in Spain.

Data obtained from wastewater analysis can provide rapid and complementary insights in illicit drug consumption at community level. Within Europe, Spain is an important country of transit of both cocaine and cannabis. The quantity of seized drugs and prevalence of their use rank Spain at the top of Europe. Hence, the implementation of a wastewater monitoring program at national level would help to get better understanding of spatial differences and trends in use of illicit drugs. In this study, a national wastewater campaign was performed for the first time to get more insight on the consumption of illicit drugs within Spain. The 13 Spanish cities monitored cover approximately 6 million inhabitants (12.8% of the Spanish population). Untreated wastewater samples were analyzed for urinary biomarkers of amphetamine, methamphetamine, MDMA, cocaine, and cannabis. In addition, weekend samples were monitored for 17 new psychoactive substances. Cannabis and cocaine are the most consumed drugs in Spain, but geographical variations showed, for instance, comparatively higher levels of methamphetamine in Barcelona and amphetamine in Bilbao, with about 1-fold higher consumption of these two substances in such metropolitan areas. For amphetamine, an enantiomeric profiling was performed in order to assure the results were due to consumption and not to illegal dumping of production residues. Furthermore, different correction factors for the excretion of cannabis were used to compare consumption estimations. All wastewater results were compared with previously reported data, national seizure data and general population survey data, were a reasonable agreement was found. Daily and yearly drug consumption were extrapolated to the entire Spanish population with due precautions because of the uncertainty associated. These data was further used to estimate the retail drug market, where for instance cocaine illicit consumption alone was calculated to contribute to 0.2-0.5% of the Spanish gross domestic product (ca. 3000-6000 million Euro/year).

Profiling cocaine residues and pyrolytic products in wastewater by mixed-mode liquid chromatography-tandem mass spectrometry.

This work provides a new analytical method for the determination of cocaine, its metabolites benzoylecgonine and cocaethylene, the pyrolytic products anhydroecgonine and anhydroecgonine methyl ester, and the pharmaceutical levamisole in wastewater. Samples were solid-phase extracted and extracts analyzed by liquid chromatography-tandem mass spectrometry using, for the first time in the illicit drug field, a stationary phase that combines reversed-phase and weak cation-exchange functionalities. The overall method performance was satisfactory, with limits of detection below 1 ng/L, relative standard deviations below 21%, and percentages of recovery between 93% and 121%. Analysis of 24-hour composite raw wastewater samples collected in Santiago de Compostela (Spain) and Brasilia (Brazil) highlighted benzoylecgonine as the compound showing the highest population-normalized mass loads (300-1000 mg/day/1000 inhabitants). In Brasilia, cocaine and levamisole loads underwent an upsurge on Sunday, indicating a high consumption, and likely a direct disposal, of cocaine powder on this day. Conversely, the pyrolytic product resulting from the smoke of crack, anhydroecgonine methyl ester, and its metabolite anhydroecgonine were relatively stable over the four days, agreeing with a non-recreational-associated use of crack.