Desarrollo de metodología analítica y estudio medioambiental de agentes antimicrobianos y drogas de abuso

La presente tesis doctoral ha abordado el estudio medioambiental de dos grandes familias de contaminantes emergentes: los agentes antimicrobianos y las drogas de abuso. Dado que ambos grupos de compuestos se introducen en el medio ambiente, fundamentalmente, a través de las aguas residuales urbanas, el principal objetivo ha sido el desarrollo de metodologías analíticas robustas, sensibles y selectivas para su determinación en esta matriz. En menor medida, se han considerado también aguas fluviales sometidas a descargas de estaciones depuradoras y, en el caso de uno de los agentes antimicrobianos más apolares, lodos procedentes de estas estaciones y sedimentos fluviales y marinos. La aplicación de las metodologías propuestas a muestras reales ha permitido su validación y ha aportado datos de distribución de estos contaminantes en el medio ambiente

Ultrasound-Assisted Extraction, Followed by Gas Chromatography–Mass Spectrometry for the Simultaneous Quantification of Ethinyl Estradiol and Drospirenone in Contraceptive Formulations

[EN]Contraceptive tablets typically contain a combination of two synthetic versions of an estrogen and a progestogen, which work together to inhibit the ovulation process. An accurate and precise quantification of these components is essential for contraceptive producers. In this study, we have developed the first gas chromatography–mass spectrometry (GC–MS) method for the simultaneous quantification of 17α-ethinyl estradiol (EE) and drospirenone (DP) in contraceptive formulations. Under the final working conditions, analytes were extracted from the solid by ultrasound-assisted extraction (15 min) in methanol. The resulting suspension was diluted in ethyl acetate, subjected to centrifugation and, finally, the supernatant was directly injected into the GC–MS system. No derivatization reagents were utilized. To correct for instrumental variations, calibration was performed using the internal standard method, with cholesterol as the internal standard. A good linearity was achieved throughout the calibration range for both EE (3–12 µg mL−1) and DP (300–1200 µg mL−1), with R2 values exceeding 0.99. Trueness, assessed in terms of percentages of recovery, was also found to be satisfactory for both analytes, with recovery rates of 106 ± 8% for EE and 93 ± 9% for DP. Furthermore, intra-day and inter-day precision studies yielded relative standard deviation values below 6% for both analytes. In terms of sensitivity, the instrumental limits of detection were 0.25 µg mL−1 for EE and 6.6 µg mL−1 for DP, and the instrumental limits of quantification 0.82 µg mL−1 for EE and 22 µg mL−1 for DP. The method was successfully applied to the analysis of contraceptive tablets from three different pharmaceutical companies. No differences were observed between the measured and the declared amount of active principle per tablet, demonstrating the applicability of the procedure. In addition, a stability study conducted on both the standards and sample extracts demonstrated that they can be stored at room temperature for a minimum period of seven days.Junta de Castilla y Leó

Wastewater-Based Epidemiology as a New Tool for Estimating Population Exposure to Phthalate Plasticizers

This study proposes the monitoring of phthalate metabolites in wastewater as a nonintrusive and economic alternative to urine analysis for estimating human exposure to phthalates. To this end, a solid-phase extraction–liquid chromatography–tandem mass spectrometry method was developed, allowing for the determination of eight phthalate metabolites in wastewater (limits of quantification between 0.5 and 32 ng L–1). The analysis of samples from the NW region of Spain showed that these substances occur in raw wastewater up to ca. 1.6 μg L–1 and in treated wastewater up to ca. 1 μg L–1. Concentrations in raw wastewater were converted into levels of exposure to six phthalate diesters. For two of them, these levels were always below the daily exposure thresholds recommended by the U.S. Environmental Protection Agency and the European Food Safety Authority. For the other four, however, estimates of exposure surpassed such a threshold (especially the toddler threshold) in some cases, highlighting the significance of the exposure to phthalates in children. Finally, concentrations in wastewater were also used to estimate metabolite concentrations in urine, providing a reasonable concordance between our results and the data obtained in two previous biomonitoring studiesThis work was financed by Spanish Ministry of Economy and Competitiveness (MINECO; project no. CTM2014-56628-C3-2-R), Xunta de Galicia (GRC2013-020 and IGM postdoctoral contract “Plan Galego de Investigación, Innovación e Crecemento 2011–2015”) and FEDER/ERDFS

Determination of human metabolites of chlorinated phosphorous flame retardants in wastewater by N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide-derivatization and gas chromatography-high resolution mass spectrometry

The analysis of wastewater for the determination of human biomarkers of exposure (human metabolites) is a non-intrusive, economic and complementary alternative to the analysis of urine in the monitoring of human exposure to chemicals of concern. This study provides the first gas chromatography-based method for the determination of three metabolites of chlorinated organophosphorous flame retardants (OPFRs: bis(2-chloroethyl) phosphate, bis(chloropropyl) phosphate and bis(1,3-dichloro-2-propyl) phosphate) in wastewater. A solid-phase extraction procedure based on the use of mixed-mode reversed-phase weak anion exchange sorbents was optimized including a fractionated elution of OPFRs and their metabolites. Analytes derivatization was investigated by comparing two silylating reagents, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide and N-methyl-N-(trimethylsilyl)trifluoroacetamide, the first one providing better results. Determination was performed by gas chromatography-high resolution mass spectrometry with a quadrupole-time-of-flight system (GC-QTOF) in order to improve selectivity. Furthermore, the use of GC-QTOF combined with the specific ion obtained from silylated metabolites (m/z 154.9924) can be exploited to screen for other phosphate ester metabolites. Under final conditions, the overall method performance was satisfactory, affording method detection limits ranging from 1.1 to 4.6 ng/L, percentages of recovery from 90% to 110%, and relative standard deviations below 13%. The analysis of composite raw wastewater samples collected over 24 h in the NW of Spain allowed to quantify, for the first time in this matrix, the metabolite bis(chloropropyl) phosphate at levels over 60 ng/LThis work was financially supported by the Spanish Agencia Estatal de Investigación (project no. CTM2017-84763-C3-2-R), the Galician Council of Culture, Education and Universities (ED431C2017/36, VC predoctoral contract, ED481A-2017/156, and IGM postdoctoral contract, Plan Galego I2C-Modalidade B, ED481D 2017/003), Gil Dávila Foundation (VC research grant) and FEDER/ERDFS

Multi-residue determination of psychoactive pharmaceuticals, illicit drugs and related metabolites in wastewater by Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry

This is the Author’s Accepted Manuscript of the following article: González-Mariño, I., Castro, V., Montes, R., Rodil, R., Lores, A., Cela, R., & Quintana, J. (2018). Multi-residue determination of psychoactive pharmaceuticals, illicit drugs and related metabolites in wastewater by ultra-high performance liquid chromatography-tandem mass spectrometry. Journal Of Chromatography A, 1569, 91-100. doi: 10.1016/j.chroma.2018.07.045 © Elsevier 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 licenseThis study presents a new multi-residue analytical method for the simultaneous determination of 38 psychoactive drugs (including benzodiazepines, antidepressants and drugs of abuse) and related metabolites in raw wastewater. Potential analyte losses during sample filtration and stability in wastewater were evaluated. Analyte losses, especially for 12 compounds, were observed during filtration, indicating a strong sorption onto the filter material. In order to overcome this effect, filtered water samples were combined with methanolic washes of the corresponding filters and the resulting solutions were solid-phase extracted on mixed-mode (reverse-phase plus cation-exchange) sorbents. Extracts were analyzed by ultra-high performance liquid chromatography-tandem mass spectrometry. Quantification was performed by the internal standard method with isotopic labeled analogs. Recovery percentages varied between 65% and 137%; method quantification limits ranged between 0.2 and 22 ng/L in ultrapure water and between 0.3 and 30 ng/L in wastewater for all the analytes but three (for which they were ∼60–80 ng/L). The analysis of 24 h-composite samples collected during one week in the city of Santiago de Compostela demonstrated the ubiquity of 31 analytes, which were positively quantified in all samples. The highest concentrations were found for some of the antidepressants, with mean and maximum levels exceeding, in some cases, the levels previously reported in literature. This fact could be related to the additional washing step of the filters using methanol, which allowed to desorb retained analytes highlighting the importance of this step during the sample preparation protocolS

Assessing Cocaine Use Patterns in the Brazilian Capital by Wastewater-Based Epidemiology

This is the postprint (accepted manuscript) version of the article published by Taylor & Francis in the International Journal of Environmental Analytical Chemistry on December, 10th 2018. Available on-line: http://www.tandfonline.com//10.1080/03067319.2018.1554743The levels of cocaine (COC) and its metabolites benzoylecgonine (BE) and cocaethylene (COE) in wastewater samples from Brasilia, Brazil, were determined using liquid chromatography coupled to hybrid quadrupole-time-of-flight mass spectrometry. The former metabolite was used to estimate cocaine consumption using a country-tuned correction factor that considers BE excretion rates for different routes of administration as well as the fraction of crack users in Brazil. A day-to-day analysis during a week indicates higher cocaine use in weekends with peaks on Sunday in both southern (7385 ± 121 mg day−1 1000 inhab−1) and northern (3566 ± 171 mg day−1 1000 inhab−1) areas of Brasilia. A significant high cocaine use was also observed in the Carnival Day of 2018 (6229 ± 219 mg day−1 1000 inhab−1). COE/BE ratios were used to assess COC and alcohol co-consumption since COE is produced during the co-consumption of both substances. Higher ratios were also observed during the weekend. However, higher ratios on Saturdays rather than Sundays may be explained by the decrease of BE excretion during the co-consumption with alcohol, which may lead to an underestimation of the cocaine use estimates on Saturdays, as well as by different patterns of co-consumption by powder and crack users, where the latter usually drink lower amounts of alcohol. Our data suggest that only 3% of cocaine was seized by the local Technical Police during the studyThis work was supported by the Federal District Research Foundation under Grant [193.000.916/ 2015]; the Spanish Agencia Estatal de Investigación under Grant [CTM2017-84763-C3-2-R]; the Galician Council of Culture, Education and Universities under Grants [ED431C2017/36]; IGM postdoctoral contract, Plan Galego I2C-Modalidade B, ED481D 2017/003; and FEDER/ERDFS

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

This is the peer reviewed version of the following article: González‐Mariño, I, Estévez‐Danta, A, Rodil, R, et al. Profiling cocaine residues and pyrolytic products in wastewater by mixed‐mode liquid chromatography–tandem mass spectrometry. Drug Test Anal. 2019; 11: 1018– 1027, which has been published in final form at https://doi.org/10.1002/dta.2590. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived VersionsThis 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 crackThis work was financially supported by Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia (ED431C2017/36 and IGM postdoctoral contract, Plan Galego I2C‐Modalidade B, ED481D 2017/003), Ministerio de Economía y Competitividad (project no. CTM2017‐84763‐C3‐2‐R), the Federal District Research Foundation (project no. 193.000.916/2015), and FEDER/ERDFS

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(-)-MDMAThis study was supported by MINECO/MICINN/AEI projects (CTM2016-81935-REDT, CTM2017-84763-C3-2-R, CTM2017-84763-C3-1-R, CTM2017-84763-C3-3-R, PID2020-117686RB-C32, PID2020-117686RB-C31, PID2020-117686RB-C33), Galician Council of Culture, Education and Universities (ED481D-2017/003, ED431C-2017/36, ED481A-2020/258 and ED431C 2021/06), cofounded by FEDER/ERDF. UJI authors acknowledge the financial support of Generalitat Valenciana (Excellence Research Group, Prometeo 2019/040). Alberto Celma acknowledges the Spanish Ministry of Economy and Competitiveness for his predoctoral Grant (BES-2016-076914). Vanessa Gutmann acknowledges the support of the ERASMUS+ program.S

ESAR-Net: a collaborative effort to expand the application of wastewater epidemiology in Spain

Resumen del trabajo presentado en el SETAC Europe 30th Annual Meeting, celebrado en modalidad virtual del 3 al 7 de mayo de 2020.Data obtained from wastewater analysis can provide rapid and complementary insights in illict drug consumption at community level. Drug use has been assessed through wastewater analysis at national level in, for example, Australia, Belgium, Finland and South Korea and has also provided annually a one week snapshot of illicit drug volumes consumed in European cities (http://www.emcdda.europa.eu/topics/ pods/waste-water-analysis). However, a wastewater monitoring program did not exist in Spain, but leading experts have formed a network (https://www.esarnet.es/) to promote wastewater-based epidemiology at national level and communicate their findings to authorities and policymakers. Within Europe, Spain is an important country of transit of both cocaine and cannabis, due to its cultural, linguistic and colonial ties to Latin America and its proximity to Morocco. The quantity of seized cocaine and cannabis and prevalence of use, locates Spain at the top of Europe. In this work, a national wastewater campaign has been performed to get more insight on the consumption of illicit drugs and NPS within Spain for the first time. Wastewater results from 14 Spanish cities were compared with previously reported data and other national indicators. The cities, located in 7 of the 17 autonomous communities, 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 to these conventional drugs, weekend samples were monitored for several new psychoactive substances (NPS) (i.e. phenethylamines and cathinones). The selected NPS are known as possible replacement of these conventional drugs or among those previously reported. Finally, enantiomeric profiling of amphetamine was performed for one city in order to assure the results were due to consumption and not illegal dumping of production residues. This demonstrates another application of wastewater-based epidemiology, which allows to identify the originof drugs in wastewater.This work has been supported by the Spanish State Research Agency (Agencia Estatal de Investigación, AEI) through the “Redes de Excelencia” programme, ESAR-Net, ref. CTM2016-81935-RED

Assessing alcohol consumption through wastewater-based epidemiology: Spain as a case study

Background In this study, an alternative and complementary method to those approaches currently used to estimate alcohol consumption by the population is described. This method, known as wastewater-based epidemiology (WBE), allows back-calculating the alcohol consumption rate in a given population from the concentrations of a selected biomarker measured in wastewater. Methods Composite (24-h) wastewater samples were collected at the inlet of 17 wastewater treatment plants located in 13 Spanish cities for seven consecutive days in 2018. The sampled area covered 12.8% of the Spanish population. Wastewater samples were analyzed to determine the concentration of ethyl sulfate, the biomarker used to back-calculate alcohol consumption. Results Alcohol consumption ranged from 4.5 to 46 mL/day/inhabitant. Differences in consumption were statistically significant among the investigated cities and between weekdays and weekends. WBE-derived estimates of alcohol consumption were comparable to those reported by its corresponding region in the Spanish National Health Survey in most cases. At the national level, comparable results were obtained between the WBE-derived annual consumption rate (5.7 ± 1.2 L ethanol per capita (aged 15+)) and that reported by the National Health Survey (4.7 L ethanol per capita (aged 15+)). Conclusions This is the largest WBE study carried out to date in Spain to estimate alcohol consumption rates. It confirms that this approach is useful for establishing spatial and temporal patterns of alcohol consumption, which could contribute to the development of health care management plans and policies. Contrary to established methods, it allows obtaining information in a fast and relatively economical wayThis work has been supported by the Spanish State Research Agency (AEI) (This work has been supported by the Spanish State Research Agency , CTM2017-84763-C3-1-R, CTM2017-84763-C3-3-R and CTM2017-84763-C3-2-R), Generalitat de Catalunya (expedient number SA-2018-780 and Consolidated Research Group 2017 SGR 01404-Water and Soil Quality Unit), and Galician Council of Culture, Education and Universities (ED481D 2017/003 and EM2014/004). Several of these projects are cofounded by the European Regional Development FundS