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The use of pesticides is often regarded as a fundamental aspect of conventional agriculture. However, these compounds have gained recognition as some of the oldest and most widely employed xenobiotic contaminants, necessitating effective strategies for human biomonitoring. In this context, a method was developed for the determination of 16 legacy organochlorine pesticides, 6 metabolites of current pesticides (2,4-D, malathion, parathion, fipronil, pyraclostrobin, cypermethrin, permethrin, cyfluthrin), and 1 triazine herbicide (atrazine) in serum. Samples were prepared with water, formic acid, acetonitrile, and ultrasound irradiation, followed by solid-phase extraction with Oasis Prime HLB. Subsequently, metabolites from current pesticides underwent derivatization using MTBSTFA with 1% TBDMSCl for analysis via gas chromatography-tandem mass spectrometry (GC-MS/MS), employing an SLB-5MS fused silica capillary column. Analytical curves were generated with limits of quantification from 0.3 to 4.0 ng.mL -1 . Accuracy ranged from 69 to 124%, and the coefficient of variation from 2 to 28%. Moreover, determining 1-(4-chlorophenyl)-1H-pyrazol-3-ol was suggested as a biomarker for pyraclostrobin biomonitoring. This analytical approach facilitated the determination of both legacy and metabolites of current pesticides in the same serum sample, presenting an interesting and cost-effective option for large cohorts, and multi-omics studies that evaluate time-dependent biomarkers in blood samples, thereby enabling biomonitoring within the same matrix. Furthermore, a proof-of-concept involving 10 volunteers demonstrated exposure to 9 pesticides at mean concentrations measured in ng mL -1 , consistent with findings from various biomonitoring initiatives., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Pharmaceuticals and personal care products (PPCPs) exhibit varying biodegradability during the acidogenic and methanogenic phases of anaerobic digestion. However, there is limited information regarding the end products generated during these processes. This work investigates the biotransformation products (BTPs) generated in a two-phase (TP) acidogenic-methanogenic (Ac-Mt) bioreactor using advanced suspect and nontarget strategies. Fourteen BTPs were confidently identified from ten parent PPCPs including carbamazepine (CBZ), naproxen (NPX), diclofenac (DCF), ibuprofen (IBU), acetaminophen (ACT), metoprolol (MTP), sulfamethoxazole (SMX), ciprofloxacin (CIP), methylparaben (MPB) and propylparaben (PPB). These BTPs were linked with oxidation reactions such as hydroxylation, demethylation and epoxidation. Their generation was related to organic acid production, since all metabolites were detected during acidogenesis, with some being subsequently consumed during methanogenesis, e.g., aminothiophenol and kynurenic acid. Another group of BTPs showed increased concentrations under methanogenic conditions, e.g., hydroxy-diclofenac and epoxy-carbamazepine. The most PPCPs showed high removal efficiencies (> 90 %) - SMX, CIP, NPX, MTP, ACT, MPB, PPB, while DCF, CBZ and IBU demonstrated higher persistence - DCF (42 %); CBZ (40 %), IBU (47 %). The phase separation of anaerobic digestion provided a deeper understanding of the biotransformation pathways of PPCPs, in addition to enhancing the biodegradability of the most persistent compounds, i.e., DCF, CBZ and IBU., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Liquid chromatography-mass spectrometry (LC-MS) has emerged as a powerful analytical technique for analyzing complex biological samples. Among various chromatographic stationary phases, porous graphitic carbon (PGC) columns have attracted significant attention due to their unique properties-such as the ability to separate both polar and non-polar compounds and their stability through all pH ranges and to high temperatures-besides the compatibility with LC-MS. This review discusses the applicability of PGC for SPE and separation in LC-MS-based analyses of human biological samples, highlighting the diverse applications of PGC-LC-MS in analyzing endogenous metabolites, pharmaceuticals, and biomarkers, such as glycans, proteins, oligosaccharides, sugar phosphates, and nucleotides. Additionally, the fundamental principles underlying PGC column chemistry and its advantages, challenges, and advances in method development are explored. This comprehensive review aims to provide researchers and practitioners with a valuable resource for understanding the capabilities and limitations of PGC columns in LC-MS-based analysis of human biological samples, thereby facilitating advancements in analytical methodologies and biomedical research., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Introduction: Pollution has emerged as a significant threat to humanity, necessitating a thorough evaluation of its impacts. As a result, various methods for human biomonitoring have been proposed as vital tools for assessing, managing, and mitigating exposure risks. Among these methods, urine stands out as the most commonly analyzed biological sample and the primary matrix for biomonitoring studies., Objectives: This review concentrates on exploring the literature concerning residual pesticide determination in urine, utilizing liquid and gas chromatography coupled with mass spectrometry, and its practical applications., Method: The examination focused on methods developed since 2010. Additionally, applications reported between 2015 and 2022 were thoroughly reviewed, utilizing Web of Science as a primary resource., Synthesis: Recent advancements in chromatography-mass spectrometry technology have significantly enhanced the development of multi-residue methods. These determinations are now capable of simultaneously detecting numerous pesticide residues from various chemical and use classes. Furthermore, these methods encompass analytes from a variety of environmental contaminants, offering a comprehensive approach to biomonitoring. These methodologies have been employed across diverse perspectives, including toxicological studies, assessing pesticide exposure in the general population, occupational exposure among farmers, pest control workers, horticulturists, and florists, as well as investigating consequences during pregnancy and childhood, neurodevelopmental impacts, and reproductive disorders., Future Directions: Such strategies were essential in examining the health risks associated with exposure to complex mixtures, including pesticides and other relevant compounds, thereby painting a broader and more accurate picture of human exposure. Moreover, the implementation of integrated strategies, involving international research initiatives and biomonitoring programs, is crucial to optimize resource utilization, enhancing efficiency in health risk assessment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Birolli, Lanças, dos Santos Neto and Silveira.)

Anaerobic digestion in two sequential phases, acidogenesis and methanogenesis, has been shown to be beneficial for enhancing the biomethane generation from wastewater. In this work, the application of glycerol (GOH) as a fermentation co-substrate during the wastewater treatment was evaluated on the biodegradation of different pharmaceuticals and personal care products (PPCPs). GOH co-digestion during acidogenesis led to a significant increase in the biodegradation of acetaminophen (from 78 to 89%), ciprofloxacin (from 25 to 46%), naproxen (from 73 to 86%), diclofenac (from 36 to 48%), ibuprofen (from 65 to 88%), metoprolol (from 45 to 59%), methylparaben (from 64 to 78%) and propylparaben (from 68 to 74%). The heterotrophic co-metabolism of PPCPs driven by glycerol was confirmed by the biodegradation kinetics, in which k bio (biodegradation kinetics constant) values increased from 0.18 to 2.11 to 0.27-3.60 L g -1 -VSS d -1 , for the operational phases without and with GOH, respectively. The assessment of metabolic pathways in each phase revealed that the prevalence of aromatic compounds degradation, metabolism of xenobiotics by cytochrome P 450 , and benzoate degradation routes during acidogenesis are key factors for the enzymatic mechanisms linked to the PPCPs co-metabolism. The phase separation of anaerobic digestion was effective in the PPCPs biodegradation, and the co-fermentation of glycerol provided an increase in the generation potential of biomethane in the system (energetic potential of 5.0 and 6.3 kJ g -1 -COD removed , without and with GOH, respectively). This study showed evidence that glycerol co-fermentation can exert a synergistic effect on the PPCPs removal during anaerobic digestion mediated by heterotrophic co-metabolism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

An automated microextraction by packed sorbent followed by liquid chromatography-tandem mass spectrometry (MEPS-LC-MS/MS) method was developed for the determination of four endocrine disruptors-parabens, benzophenones, and synthetic phenolic antioxidants-in wastewater samples. The method utilizes a lab-made repackable MEPS device and a multi-syringe robotic platform that provides flexibility to test small quantities (2 mg) of multiple extraction phases and enables high-throughput capabilities for efficient method development. The overall performance of the MEPS procedure, including the investigation of influencing variables and the optimization of operational parameters for the robotic platform, was comprehensively studied through univariate and multivariate experiments. Under optimized conditions, the target analytes were effectively extracted from a small sample volume of 1.5 mL, with competitive detectability and analytical confidence. The limits of detection ranged from 0.15 to 0.30 ng L -1 , and the intra-day and inter-day relative standard deviations were between 3 and 21%. The method's applicability was successfully demonstrated by determining methylparaben, propylparaben, butylated hydroxyanisole, and oxybenzone in wastewater samples collected from the São Carlos (SP, Brazil) river. Overall, the developed method proved to be a fast, sensitive, reliable, and environmentally friendly analytical tool for water quality monitoring., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Preparative liquid chromatography is a technique for separating complex samples or isolating pure compounds from complex extracts. It involves eluting samples through a packed column and selectively collecting or isolating the separated bands in a sequence of fractions. Depending on the column length and the sample complexity, a large number of fractions may be obtained, making fraction collection a laborious and time-consuming process. Manual fraction collection is also tedious, error-prone, less reproducible, and susceptible to contamination. Several commercial and lab-made solutions are available for automated fraction collection, but most systems do not synchronize with the instrument detector and collect fractions at fixed volumes or time intervals. We have assembled a low-cost Arduino-based smart fraction collector that can record the signal from the UV-vis detector of the chromatography instrument and enable the automated selective collection of the targeted bands. The system consists of a robot equipped with position sensors and a 3-way solenoid valve that switches the column effluent between the waste or collection positions. By proper programming, an Arduino board records the detector response and actuates the solenoid valve, the position sensors, and the stepper motors to collect the target chromatographic bands., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 Published by Elsevier Ltd.)

Sample preparation frequently is considered the most critical stage of the analytical workflow. It affects the analytical throughput and costs; moreover, it is the primary source of error and possible sample contamination. To increase efficiency, productivity, and reliability, while minimizing costs and environmental impacts, miniaturization and automation of sample preparation are necessary. Nowadays, several types of liquid-phase and solid-phase microextractions are available, as well as different automatization strategies. Thus, this review summarizes recent developments in automated microextractions coupled with liquid chromatography, from 2016 to 2022. Therefore, outstanding technologies and their main outcomes, as well as miniaturization and automation of sample preparation, are critically analyzed. Focus is given to main microextraction automation strategies, such as flow techniques, robotic systems, and column-switching approaches, reviewing their applications to the determination of small organic molecules in biological, environmental, and food/beverage samples., (© 2023 Wiley-VCH GmbH.)

The organic matter bioconversion into methane during anaerobic digestion (AD) comprises different steps, the acidogenic and methanogenic phases being clearly distinct in terms of metabolic activities. In this work, new configurations of anaerobic fixed bed biofilm reactors (AFBBR) were operated under conventional methanogenic conditions (single phase - SP-AFBBR, M 1 R), and in a sequential two-phase system, acidogenic reactor followed by methanogenic reactor (TP-AFBBR, AcR + M 2 R), in order to verify the impact of the AD phase separation on the overall system performance in operational, kinetics and microbiological aspects. The results indicated that feeding the methanogenic reactor with the acidogenic effluent stream provided a shorter operating start-up period (11 and 32 days for SP and TP-AFBBR, respectively), a greater alkalinity generation (0.14 and 0.41 g-CaCO 3 ·g-COD removed -1 for M 1 R and M 2 R, respectively), and the optimization of biomethane production (methane yield of 95 and 154 N-mLCH 4 ·g-COD removed -1 for M 1 R and M 2 R, respectively). The COD removal kinetics was also favored in the TP-AFBBR (k 1-COD  = 1.4 and 2.9 h -1 for M 1 R and M 2 R, respectively), since the soluble fermentation products were readily bioavailable to the biomass in the reactor. Hydrogenotrophic methanogenesis was the predominant pathway in the M 2 R, while the Methanosaeta-driven acetoclastic pathway predominated in the M 1 R. The greater diversity of Bacteria and Archaea in M 2 R denotes a better balance between the species that degrade volatile organic acids from AcR (i.e. Syntrophorhabdus, Syntrophus and Syntrophobacter) and the hydrogenotrophic methanogens (Methanoregula, Methanolinea and Methanospirillum) that consume the biodegradation products. The estimated bioenergy generation potential (range of 0.39-0.64 kWh·m -3 -sewage considering the COD removed) for full-scale TP-sewage treatment plants evidences the feasibility of energetic recovery in the domestic sewage anaerobic treatment., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Dynamic single-drop microextraction (SDME) was automatized employing an Arduino-based lab-made Cartesian robot and implemented to determine parabens in wastewater samples in combination with liquid chromatography-tandem mass spectrometry. A dedicated Arduino sketch controls the auto-performance of all the stages of the SDME process, including syringe filling, drop exposition, solvent recycling, and extract collection. Univariate and multivariate experiments investigated the main variables affecting the SDME performance, including robot-dependent and additional operational parameters. Under selected conditions, limit of detections were established at 0.3 µg/L for all the analytes, and the method provided linear responses in the range between 0.6 and 10 µg/L, with adequate reproducibility, measured as intraday relative standard deviations (RSDs) between 5.54% and 17.94%, (n = 6), and inter-days RSDs between 8.97% and 16.49% (n = 9). The robot-assisted technique eased the control of dynamic SDME, making the process more feasible, robust, and reliable so that the developed setup demonstrated to be a competitive strategy for the automated extraction of organic pollutants from water samples., (© 2022 Wiley-VCH GmbH.)

Cannabinoids are pharmacologically active compounds present in cannabis plants, which have become important research topics in the modern toxicological and medical research fields. Not only is cannabis the most used drug globally, but also cannabinoids have a growing use to treat a series of diseases. Therefore, new, fast, and efficient analytical methods for analyzing these substances in different matrices are demanded. This study developed a new packed-in-tube solid-phase microextraction (IT-SPME) method coupled to liquid chromatography with tandem mass spectrometry (LC-MS/MS), for the automated microextraction of seven cannabinoids from human urine. Packed IT-SPME microcolumns were prepared in (508 µm i.d. × 50 mm) stainless-steel hardware; each one required only 12 mg of sorbent phase. Different sorbents were evaluated; fractional factorial design 2 4-1 and a central composite design were employed for microextraction optimization. Under optimized conditions, the developed method was a fast and straightforward approach. Only 250 µl of urine sample was needed, and no hydrolysis was required. The sample pretreatment included only dilution and centrifugation steps (8 min), whereas the complete IT-SPME-LC-MS/MS method took another 12 min, with a sample throughput of 3 samples h -1 . The developed method presented adequate precision, accuracy and linearity; R 2 values ranged from 0.990 to 0.997, in the range of 10-1000 ng ml -1 . The lower limits of quantification varied from 10 to 25 ng ml -1 . Finally, the method was successfully applied to analyze 20 actual urine samples, and the IT-SPME microcolumn was reused over 150 times., (© 2022 Wiley-VCH GmbH.)

An on-line solid phase extraction using a lab-made restricted access media (RAM) was developed as sample preparation procedure for determination of the pharmaceutical compounds caffeine (CAF), carbamazepine (CBZ), norfloxacin (NOR), ciprofloxacin (CIP), fluoxetine (FLX) and venlafaxine in wastewater treatment plant samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is suitable for use in routine of analysis, avoiding cross-contamination and requiring only a small sample volume (50 µL), with minimal handling. The method was validated according to international guidelines. The chromatographic efficiency was evaluated using peak resolution and asymmetry parameters. Carryover was also evaluated, in order to ensure reliability of the analysis and the ability to reuse the cartridge. Satisfactory linearity (r 2  > 0.99) was obtained for all the compounds. The intra- and inter-day precision values were lower than 5.79 and 14.1%, respectively. The limits of detection ranged from 0.01 to 3 µg L -1 and the limits of quantification were from 0.1 to 5 µg L -1 . The method was applied to 20 environmental wastewater samples, with caffeine being the most widely detected compound, at the highest concentration of 392 µg L -1 , while other compounds were detected in fewer samples at lower concentrations (up to 9.60 µg L -1 ). The lab-made modification is a cheaper option for on-line sample preparation, compared to commercially available on-line SPE cartridges and RAM columns. Moreover, a high-throughput procedure was achieved, with an analysis time of 16 min including sample preparation and chromatographic separation. The same RAM column was applied over 200 injections including method optimization, validation and application in wastewater samples without loss of analytical response., (Copyright © 2021 Elsevier B.V. All rights reserved.)

New sanitation systems have been developed to treat, recover energy and nutrients, and permit reuse processes at the source of generation, minimizing water use and flow segregation. Thus, this study was carried out with the objective of evaluating the potential of hybrid constructed wetlands in the removal of organic matter, nutrients, pathogenic microorganisms, and 12 antibiotics from blackwater previously treated by an upflow anaerobic sludge blanket reactor. A hybrid system of constructed wetlands was used, comprised of a horizontal subsurface flow constructed wetland with a total volume of 0.60 m 3 followed by a vertical subsurface flow constructed wetland with a total volume of 0.20 m 3 . Three different hydraulic retention times were comparatively tested (1.0, 2.0, and 3.0 days for the horizontal subsurface flow constructed wetland, and 1.1, 0.9, and 0.4 days for the vertical subsurface flow constructed wetland) in four distinct stages. The plant species used was Canna x generalis. The results from this study demonstrate the potential of constructed wetlands as a suitable technology for post-treatment of segregated domestic wastewater (anaerobically-digested blackwater). Efficient reduction of COD, BOD 5 , total nitrogen, and total phosphorus (74, 93, 50, and 61%, respectively) was achieved, with a hydraulic retention time of 3.0 and 1.1 days for horizontal and vertical subsurface flow constructed wetland, respectively (stage IV). The presence of ciprofloxacin was confirmed by chromatographic and mass spectrometric analysis in an average concentration of 442.6 ng.L -1  at the inflow of the horizontal subsurface flow constructed wetland, but was not observed at the outflow., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

An original, selective and automated method, for the microextraction by packed sorbent (MEPS) of Δ9-tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (THC-OH), and 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) from human urine, was developed by using (i) a catechin-molded molecularly imprinted polymer (MIP), (ii) a new lab-made MEPS device easily repackable with any commercial or lab-made sorbent, and (iii) a lab-made multi syringe autosampler. Analyses were performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the developed method proved to be precise, accurate and showed good linearity. Determination coefficients ranged from 0.96 to 0.99, in the range of 5-250 ng mL -1 . Limits of detection and quantification ranged between 1.0 and 5.0 ng mL -1 and 5.0 and 20.0 ng mL -1 . The method was successfully applied in the analysis of real urine samples. The same packed syringe was effectively used over 90 consecutive extractions without carry-over effects., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Antibiotic compounds, notably sulfamethoxazole (SMX) and ciprofloxacin (CIP), are ubiquitous emerging contaminants (ECs), which are often found in domestic sewage. They are associated with the development of antimicrobial resistance. Operational parameters, e.g. organic loading rate (OLR), hydraulic retention time (HRT) and sludge retention time, may influence EC biodegradation in wastewater treatment plants. This study assessed the impact of the OLR variation on the biodegradation of CIP and SMX, applying two configurations of anaerobic fixed bed reactors: anaerobic packed bed biofilm reactor (APBBR) and anaerobic structured bed biofilm reactor (ASBBR). A significant reduction in the biodegradation of SMX (APBBR: 93-69%; ASBBR: 94-81%) and CIP (APBBR: 85-66%; ASBBR: 85-64%) was observed increasing OLR from 0.6 to 2.0 kgCOD m -3 d -1 . The decrease in the HRT from 12 to 4 h resulted in higher liquid-phase mass transfer coefficient (APBBR: k s from 0.01 to 0.05 cm h -1 ; ASBBR: k s from 0.07 to 0.24 cm h -1 ), but this was not enough to overcome the decrease in the antibiotic-biomass contact time on biofilm, thus reducing the bioreactors' performance. The ASBBR favored biomethane production (from 7 to 17 mLCH 4 g -1 VSS L -1 d -1 ) and biodegradation kinetics (k bio from 1.7 to 4.2 and for SMX and from 2.1 to 4.8 L g -1 VSS d -1 for CIP) due to the higher relative abundance of the archaea community in the biofilm and the lower liquid-phase mass transfer resistance in the structured bed. CIP and SMX cometabolic biodegradation was associated to the hydrogenotrophic methanogenesis (mainly Methanobacterium genus) in co-culture with fermentative bacteria (notably the genera Clostridium, Bacillus, Lactivibrio, Syntrophobacter and Syntrophorhabdus). The anaerobic fixed bed biofilm reactors proved to be highly efficient in biodegrading the antibiotics, preventing them from spreading to the environment., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

By proper design of an innovative extraction device, a lab-made multipurpose autosampler was exploited in the automated performance of the dynamic large drops based microextraction. The pluses of this new analytical strategy were demonstrated in the determination of sulfonamides and fluoroquinolones in surface water samples, by direct immersion single drop microextraction (SDME) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis. Operational autosampler features and critical experimental factors influencing SDME, including the extraction mode (static or dynamic), extraction, stirring rate, salt addition, drop size, number of cycles and drop exposition time, were comprehensively investigated using both univariate and multivariate optimization. The lab-made autosampler allowed to performance challenging dynamic and static large drop based SDMEs in an automated and effortless way and with minimal requirements of hardware and software. Large stable drops provided high surface area, enhancing the phase ratio and in consequence increasing the analytes uptake. The best extraction efficiencies were obtained as a result of the synergic interaction between the use of large drops and the automated dynamic mode of extraction. The developed method proved to be a reliable, sensitive, and robust analytical tool, with intraday RSDs ranging between 4.0 and 7.6% (n = 6), and interday RSDs between 4.8 and 9.3% (n = 6), and, LOD and LOQ in the range of 15-50 and 35-100 ng L -1 , respectively., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The present paper describes an original method for the online preconcentration and analysis of ring-substituted amphetamines in urine samples, used on the integration of robot-assisted hollow fiber liquid-phase microextraction (HF-LPME), high-performance liquid chromatography (HPLC), and fluorescence detection (FLD). A lab-made autosampler, actuating a 100-μL syringe and equipped with a three-way solenoid microvalve, allowed the acceptor phase to flow through and be withdrawn from the lumen fiber, enabling the automated online transference of the enriched acceptor phase for chromatographic analysis, through a six-port switching valve. The developed online HF-LPME-LC/FLD method demonstrated high analytical throughput and confidence, facilitating the efficient extraction and determination of the target analytes, with minimal solvent consumption and sample manipulation, in a straightforward way. Sample cleanup, analyte uptake, and analysis were carried out in 14.5 min. Under optimal conditions, automated online HF-LPME showed excellent linearity, precision, and trueness, obtaining intraday RSDs between 2.9 and 9.2% (n = 6) and interday RSDs between 5.3 and 9.3% (n = 6). Enrichment factors (EFs) ranged between 14.2 and 15.7, extraction recoveries (ERs) ranged between 17.7 and 19.5%, and the limits of detection (S/N = 3) were 2.0, 3.0, and 3.0 μg L -1 for MDA, MDMA, and MDEA, respectively. The method proved to be an effortless, rapid, reliable, and environment-friendly approach for the determination of drug abuse in urine samples. Graphical abstract.

Metabolomics studies of the early-life exposome often use maternal urine specimens to investigate critical developmental windows, including the periconceptional period and early pregnancy. During these windows changes in kidney function can impact urine concentration. This makes accounting for differential urinary dilution across samples challenging. Because there is no consensus on the ideal normalization approach for urinary metabolomics data, this study's objective was to determine the optimal post-analytical normalization approach for untargeted metabolomics analysis from a periconceptional cohort of 45 women. Urine samples consisted of 90 paired pre- and post-implantation samples. After untargeted mass spectrometry-based metabolomics analysis, we systematically compared the performance of three common approaches to adjust for urinary dilution-creatinine adjustment, specific gravity adjustment, and probabilistic quotient normalization (PQN)-using unsupervised principal components analysis, relative standard deviation (RSD) of pooled quality control samples, and orthogonal partial least-squares discriminant analysis (OPLS-DA). Results showed that creatinine adjustment is not a reliable approach to normalize urinary periconceptional metabolomics data. Either specific gravity or PQN are more reliable methods to adjust for urinary concentration, with tighter quality control sample clustering, lower RSD, and better OPLS-DA performance compared to creatinine adjustment. These findings have implications for metabolomics analyses on urine samples taken around the time of conception and in contexts where kidney function may be altered.

This study assessed the applicability of fixed bed bioreactors in two configurations - anaerobic structured bed reactor (ASBR) and anaerobic packed bed reactor (APBR) - in the removal of Sulfamethoxazole (SMX) and Ciprofloxacin (CIP), two antibiotics frequently detected in sanitary sewage. The problem of these pharmaceuticals as emerging contaminants in conventional sewage treatment systems is mainly because they encourage the development and spread of resistance genes in bacteria. Both reactors had similar performances, and the antibiotics were highly removed - APBR: 85 ± 10% for SMX and 81 ± 16% for CIP; ASBR: 83 ± 12% for SMX and 81 ± 15% for CIP. The ASBR showed to be potentially more feasible in operating and economic terms compared to the APBR, as the former presents a smaller amount of support material in the bed. SMX was completely biotransformed, while the influence of the sorption mechanism was observed for CIP, as its presence was detected sorbed onto biomass throughout the reaction bed of the reactors, with a partition coefficient (log K D ) of around 2.8 L·kg -1 TSS. The degradation kinetics of the pharmaceuticals were fitted using a first-order kinetic model, whereby the reactors behaved as plug flow ones, indicating the possibility of optimizing the operation for a hydraulic retention time of 6 h. The removal kinetics was more favorable for CIP (higher apparent constant kinetic - k CIP app  > k SMX app ), since its biodegradation is linked to the biomass, which is more concentrated in the bed bottom layer. The experimental results showed the potential of anaerobic fixed bed reactors in removing environmentally relevant concentrations of SMX and CIP found in sewage., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A high-throughput and innovative setup has been developed to automate the online integration of single drop microextraction (SDME), liquid chromatography (LC) and high-resolution mass spectrometry (QqToF). SDME and LC were online hyphenated for the first time. SDME was carried out by a lab-made cartesian robot actuating a 100 μL syringe, equipped with a three-way solenoid microvalve that allowed the online transference of the enriched extract to the chromatographic system, through a six-port switching valve. The complete method, including the synchronized robot action, valves, and the analytical instruments, was controlled by an Arduino Mega board. The merits of the proposed setup were demonstrated by the triazines determination in coconut water samples. The most relevant extraction parameters, such as drop size, exposure time, stirring effect, salt addition and pH were systematically investigated. Under optimized conditions (60 μL drop volume and 10 min extraction time), the LC-UV enrichment factors (EF) and the extraction recoveries (ER) ranged between 15.2-18.4 and 11.4-13.8%, respectively. Using the SDME-LC-MS setup, the linear range, detection limit (S/N = 3) and precision (RSD, n = 6 at 0.25 μg L -1 level of concentration) were 0.25-25 μg L -1 , 0.10 μg L -1 and 16.8% for simazine; 0.25-25 μg L -1 , 0.05 μg L -1 and 14.7% for atrazine; and 0.25-25 μg L -1 , 0.05 μg L -1 and 18.5% for propazine, respectively. Although none of the analytes were detected in the evaluated commercial samples, the results indicate that the proposed online SDME-LC setup is a competitive analytical strategy for the determination of target organic compounds in complex matrices., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sulfamethazine (SMZ) is an antibiotic from sulfonamides class widely used in veterinary medicine and reported in wastewater and sewage. Thus, it is essential to study technologies to reduce SMZ present in the aquatic environment. Anaerobic bioreactors are a low-cost technology applied for wastewater treatment. The objective of this paper is to study kinetics parameters related to SMZ removal using a horizontal flow-anaerobic immobilized biomass reactor (HAIB) and to evaluate its transformation products formed during this treatment. The bioreactor was operated at mesophilic condition with a hydraulic retention time of 12 h. The removal of SMZ was evaluated at three different concentrations: 200 ng L -1 (phase I), 400 ng l -1 (phase II) and 600 ng L -1 (phase III). The apparent first-order removal constant obtained for chemical oxygen demand was 0.885 ± 0.094 h -1 while SMZ showed a removal constant of 0.356 h -1 . SMZ was removed with an efficiency of 56.0 ± 13.0 % (phase I); 62.0 ± 12.0 % (phase II) and 62.0 ± 6.00 % (phase III). Seven transformation products were detected and one of these with m/z 233 is reported for the first-time. The HAIB bioreactor has a potential to assist in wastewater treatment to remove contaminants at ng L -1 concentration level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The use of anaerobic biomass attached to a support has been recently presented as a good prospect in the treatment of wastewater containing recalcitrant compounds, such as sulfamethazine (SMZ). SMZ has been found in swine wastewater and sewage treatment plants, which motivates assessing their degradation by new wastewater treatment technologies. Thus, this paper describes the use of a continuous fixed structured bed bioreactor for the purpose of evaluating SMZ removal kinetics present in lab-made wastewater. The analysis of SMZ used online solid-phase extraction coupled to liquid chromatography/tandem mass spectrometry (SPE online-LC-MS/MS). Chemical oxygen demand (COD) was also monitored to evaluate the organic matter removal. The bioreactor was operated under mesophilic conditions (30 ∘ C), with a hydraulic retention time of 24 h. In order to evaluate SMZ removal, four different concentration levels were studied: 200, 400, 600, and 800 ng L -1 . COD removal efficiency obtained for filtered effluent kept at 91.01% and there was no interference due to the increase of SMZ concentration. For SMZ, the removal efficiencies were of 52.8 ± 12.1 % for 200 ng L -1 concentration level; 55.0 ± 8.15 % for 400 ng L -1 ; 53.0 ± 6.14 % for 600 ng L -1 , and 48.8 ± 5.44 % for 800 ng L -1 . COD removal kinetics presented a first-order apparent removal rate constant ( k app ) of 0.281 ± 0.0295  h -1 . SMZ also showed a first-order apparent removal rate constant of 0.158 ± 0.0093  h -1 for the following concentrations levels: 200, 400, 600, and 800 ng L -1 .

In this work, the dispersive liquid-liquid microextraction technique based on the solidification of the organic phase (DLLME-SFO) has been automated for the first time. DLLME-SFO is automated by hyphenating a sequential injection analysis (SIA) system with a custom-made robotic phase separator. Automated in-syringe DLLME is followed by phase separation in a 3D printed device integrating a Peltier cell set, mounted on a multi-axis robotic arm. The combined action of the flow system and the robotic arm is controlled by a single software package, enabling the solidification/melting and collection of the organic phase for further analyte quantification. As proof-of-concept, automated DLLME-SFO was applied to the extraction of parabens followed by separation using liquid chromatography, obtaining LODs between 0.3 and 1.3 µg L -1 (4 mL of sample extracted in 1 mL of 1-dodecanol: MeOH, 15:85, v-v). The method showed a high reproducibility, obtaining intraday RSDs between 4.6% and 5.8% (n = 6), and interday RSDs between 5.6% and 8.6% (n = 6). The developed method was evaluated for the determination of parabens in water, urine, saliva, and personal care products., (Copyright © 2018 Elsevier B.V. All rights reserved.)

To achieve a clearer understanding of the mechanisms responsible for neutrophil dysfunction recently described in dogs with chronic renal failure (CRF), the plasma concentrations of free p-cresol in healthy dogs (n = 20) and those with CRF (n = 20) were compared. The degree of correlation was determined between plasma levels of p-cresol and markers of oxidative stress and function of neutrophils in these dogs. The effect of this compound on oxidative metabolism and apoptosis was assessed in neutrophils isolated from 16 healthy dogs incubated in RPMI 1640 supplemented with p-cresol (0.405 mg/L) and compared with medium supplemented with uremic plasma (50%). To achieve this, the plasma concentration of p-cresol was quantified by liquid phase high-performance liquid chromatography. The neutrophil oxidative metabolism was determined using the probes hydroethidine and 2',7'-dichlorofluorescein diacetate and apoptosis was measured using Annexin V-PE by capillary flow cytometry. Compared with the healthy dogs, uremic dogs presented higher concentrations of free p-cresol, greater oxidative stress, and neutrophils primed for accelerated apoptosis. The free p-cresol induced in neutrophils from healthy dogs increased apoptosis and decreased reactive oxygen species production. We conclude that the health status presented during uremia concomitant with the increase in plasma free p-cresol can contribute to the presence of immunosuppression in dogs with CRF., (Copyright © 2015 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

The antibiotics sulfamethoxazole (SMTX) and ciprofloxacin (CIP) are commonly used in human and veterinary medicine, which explains their occurrence in wastewater. Anaerobic reactors are low-cost, simple and suitable technology to wastewater treatment, but there is a lack of studies related to the removal efficiency of antibiotics. To overcome this knowledge gap, the objective of this study was to evaluate the removal kinetics of SMTX and CIP using a horizontal-flow anaerobic immobilized biomass reactor. Two different concentrations were evaluated, for SMTX 20 and 40 μg L(-1); for CIP 2.0 and 5.0 μg L(-1). The affluent and effluent analysis was carried out in liquid chromatography/tandem mass spectrometry (LC-MS/MS) with the sample preparation procedure using an off-line solid-phase extraction. This method was developed, validated and successfully applied for monitoring the affluent and effluent samples. The removal efficiency found for both antibiotics at the two concentrations studied was 97%. Chemical oxygen demand (COD) exhibited kinetic constants that were different from that observed for the antibiotics, indicating the absence of co-metabolism. Also, though the antibiotic concentration was increased, there was no inhibitory effect in the removal of COD and antibiotics.

Molecularly imprinting polymers (MIPs) can be modified with external layers in order to obtain restricted access molecularly imprinted polymers (RAMIPs) able to exclude macromolecules and retain low weight compounds. These modifications have been frequently achieved using hydrophilic monomers, chemically bound on the MIP surface. Recently, our group proposed a new biocompatible RAMIP based on the formation of a bovine serum albumin coating on the surface of MIP particles. This material has been used to extract drugs directly from untreated human plasma samples, but its physicochemical evaluation has not been carried out yet, mainly in comparison with RAMIPs obtained by hydrophilic monomers. Thus, we proposed in this paper a comparative study involving the surface composition, microscopic aspect, selectivity, binding kinetics, adsorption and macromolecule elimination ability of these different materials. We concluded that the synthesis procedure influences the size and shape of particles and that hydrophilic co-monomer addition as well as coating with BSA do not alter the chemical recognition ability of the material. The difference between imprinted and non-imprinted polymers' adsorption was evident (suggesting that imprinted polymers have a better capacity to bind the template than the non-imprinted ones). The Langmuir model presents the best fit to describe the materials' adsorption profile. The polymer covered with hydrophilic monomers presented the best adsorption for the template in an aqueous medium, probably due to a hydrophilic layer on its surface. We also concluded that an association of the hydrophilic monomers with the bovine serum albumin coating is important to obtain materials with higher capacity of macromolecule exclusion.

This study presents a column-switching solid-phase extraction online-coupled to a liquid chromatography/tandem mass spectrometry (SPE-LC-MS/MS) method for simultaneous analysis of 12 antibiotics (7 sulfonamides and 5 fluoroquinolones) and caffeine detected in the sewage and effluent of a pilot anaerobic reactor used in sewage treatment. After acidification and filtration, the samples were directly injected into a simple and conventional LC system. Backflush and foreflush modes were compared based on the theoretical plates and peak asymmetry observed. The method was tested in terms of detection (MDL) and quantification limit (MQL), linearity, relative recovery, and precision intra- and inter-day in lab-made sewage samples. The method presented suitable figures of merit in terms of detection, varying from 8.00 × 10(-5) to 6.00 × 10(-2) ng (0.800 up to 600 ng L(-1); caffeine) with direct injection volume of only 100 μL and 13 min of total analysis time (sample preparation and chromatographic run). When the method was applied in the analysis of sewage and effluent of the anaerobic reactor (n = 15), six antibiotics and caffeine were detected in concentrations ranging from 0.018 to 1097 μg L(-1). To guarantee a reliable quantification, standard addition was used to overcome the matrix effect.

Leaf-cutter ants use plant matter to culture the obligate mutualistic basidiomycete Leucoagaricus gongylophorus. This fungus mediates ant nutrition on plant resources. Furthermore, other microbes living in the fungus garden might also contribute to plant digestion. The fungus garden comprises a young sector with recently incorporated leaf fragments and an old sector with partially digested plant matter. Here, we show that the young and old sectors of the grass-cutter Atta bisphaerica fungus garden operate as a biphasic solid-state mixed fermenting system. An initial plant digestion phase occurred in the young sector in the fungus garden periphery, with prevailing hemicellulose and starch degradation into arabinose, mannose, xylose, and glucose. These products support fast microbial growth but were mostly converted into four polyols. Three polyols, mannitol, arabitol, and inositol, were secreted by L. gongylophorus, and a fourth polyol, sorbitol, was likely secreted by another, unidentified, microbe. A second plant digestion phase occurred in the old sector, located in the fungus garden core, comprising stocks of microbial biomass growing slowly on monosaccharides and polyols. This biphasic operation was efficient in mediating symbiotic nutrition on plant matter: the microbes, accounting for 4% of the fungus garden biomass, converted plant matter biomass into monosaccharides and polyols, which were completely consumed by the resident ants and microbes. However, when consumption was inhibited through laboratory manipulation, most of the plant polysaccharides were degraded, products rapidly accumulated, and yields could be preferentially switched between polyols and monosaccharides. This feature might be useful in biotechnology., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Molecularly imprinted polymers for the determination of triazines were synthesized by precipitation using atrazine as template, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker, and 2,2'-azobisisobutrynitrile as initiator. The polymers were characterized by infrared spectroscopy and scanning electron microscopy and packed in a device for microextraction by packed sorbent aiming for the preconcentration/cleanup of herbicides, such as atrazine, simazine, simetryn, ametryn, and terbutryn in corn samples. Liquid chromatography coupled with time-of-flight mass spectrometry was used for the separation and determination of the herbicides. The selectivity coefficient of molecularly imprinted polymers was compared with that of nonimprinted polymer for the binary mixtures of atrazine/propanil and atrazine/picloram, and the values obtained were 15.6 and 2.96, respectively. The analytical curve ranged from 10 to 80 μg/kg (r = 0.989) and the limits of detection and quantification in the corn matrices were 3.3 and 10 μg/kg, respectively. Intra- and interday precisions were < 14.8% and accuracy was better than 90.9% for all herbicides. Polymer synthesis was successfully applied to the cleanup and preconcentration of triazines from fortified corn samples with 91.1-109.1% of recovery., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Analysis of several emerging contaminants (steroids, caffeine and methylparaben) in water using automated solid-phase microextraction with comprehensive two dimensional gas chromatography coupled to time of flight mass spectrometry (SPME-GCxGC-ToF/MS) is presented. Experimental design was used to determine the best SPME extraction conditions and the steroids were not derivatized prior to injection. SPME-GCxGC-ToF/MS provided linear ranges from 0.6 to 1200μgL(-1) and limits of detection and quantitation from 0.02 to 100μgL(-1). A series of river water samples obtained locally were subjected to analysis. SPME-GCxGC-ToF/MS is readily automated, straightforward and competitive with other methods for low level analysis of emerging contaminants., (Copyright © 2013 Elsevier B.V. All rights reserved.)