Development and validation of an ultrafast method of quantification of rivaroxaban in human serum using laser diode thermal desorption coupled to triple quadrupole mass spectrometry.

RATIONALE: Rivaroxaban is an anticoagulant prescribed to patients who are at risk of medical conditions such as deep-vein thrombosis, pulmonary embolisms, and strokes caused by blood clots. The administration of this drug is monitored to adjust the dosage and evaluate patients' blood concentration. Rapid quantification of this drug in plasma could make it possible to ensure that the dose present in the blood of patients does not represent a danger for the medical intervention to be carried out. METHODS: Liquid chromatography-tandem mass spectrometry is usually employed to quantify rivaroxaban in blood, plasma, and serum. Here, an alternative method of analysis based on laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS) was developed and comprehensively validated. This new method allows the quantification of rivaroxaban in less than 13 s from sample to sample. The extraction of rivaroxaban in human serum was done by a salting-out liquid-liquid extraction with acetonitrile and a saturated sodium chloride solution. RESULTS: The proposed method allows the quantification of rivaroxaban in less than 13 s from sample to sample. During validation, all criteria were respected. The accuracy was <15% of the nominal value, the precision was <15%CV, and the recovery was ≥89.9%. There were no observed carryover or matrix effects. Analysis of the extracted samples established the stability of dry (24 h) and wet samples (1 week) when samples cannot be analyzed immediately, a considerable advantage in a clinical setting. CONCLUSIONS: This method improves sample throughput by more than 1200% compared to liquid chromatography-tandem mass spectrometry methods of analysis of rivaroxaban and decreases analysis costs by reducing solvent consumption and instrument time.

Trace organic contaminants in lake waters: Occurrence and environmental risk assessment at the national scale in Canada.

Numerous contaminants are produced and used daily, a significant fraction ultimately finding their way into natural waters. However, data on their distribution in lakes is lacking. To address this gap, the presence of 54 trace organic contaminants (TrOCs), representative of various human activities, was investigated in the surface water of 290 lakes across Canada. These lakes ranged from remote to highly impacted by human activities. In 88% of the sampled lakes, contaminants were detected, with up to 28 detections in a single lake. The compounds most frequently encountered were atrazine, cotinine, and deethylatrazine, each of which was present in more than a third of the lakes. The range of detected concentrations was from 0.23 ng/L to about 2200 ng/L for individual compounds, while the maximum cumulative concentration exceeded 8100 ng/L in a single lake. A risk assessment based on effect concentrations for three aquatic species (Pimephales promelas, Daphnia magna, and Tetrahymena pyriformis) was conducted, revealing that 6% of lakes exhibited a high potential risk for at least one species. In 59% of lakes, some contaminants with potential sub-lethal effects were detected, with the detection of up to 17 TrOCs with potential impacts. The results of this work provide the first reference point for monitoring the evolution of contamination in Canadian lakes by TrOCs. They demonstrate that a high proportion of the sampled lakes bear an environmentally relevant anthropogenic chemical footprint.

Are 20-hydroxyecdysone and related genes potential biomarkers of sublethal exposure to lipid-altering contaminants?

In Daphnia magna, 20-hydroecdysone (20E) is the main molting hormone and its metabolism is of interest to identify new biomarkers of exposure to contaminants. The present study aimed to (i) assess baseline levels of 20E and transcription levels of four related-genes (shade, neverland, ultraspiracle, and ecdysteroid receptor); and (ii) evaluate effects in D. magna after 21 days of exposure to fenarimol (anti-ecdysteroid) and a mixture of gemfibrozil and clofibric acid (lipid-lowering drugs) at sublethal concentrations. Endpoints included transcription of the target genes and quantification of 20E, mortality, and reproduction of daphnids. Baseline results showed that average responses were relatively similar and did not vary more than 2-fold. However, intra-day variation was generally high and could be explained by sampling individuals with slightly different stages of their development. Exposure tests indicated a significant decrease in daphnid reproduction following chronic exposure to a concentration of 565 µg/L of fenarimol. However, no difference was observed between the control and exposed groups for any of the investigated genes, nor for the levels of 20E after 21 days of exposure. Following exposition to gemfibrozil and clofibric acid at 1 µg/L, no changes were observed for the measured parameters. These results suggest that changes in transcription levels of the target genes and concentrations of 20E may not be sensitive endpoints that can be used as biomarkers of sublethal exposure to the target compounds in D. magna. Measuring multiple time points instead of a single measure as well as additional molecular endpoints obtained from transcriptomic and metabolomic studies could afford more insights on the changes occurring in exposed daphnids to lipid-altering compounds and identify efficient biomarkers of sublethal exposure.

Characterization of laccases from Trametes hirsuta in the context of bioremediation of wastewater treatment plant effluent.

The bioremediation of pharmaceutical compounds contained in wastewater, in an ecological and sustainable way, is possible via the oxidative action of fungal laccases. The discovery of new fungal laccases with unique physico-chemical characteristics pushes researchers to identify suitable laccases for specific applications. The aim of this study is to purify and characterize laccase isoenzymes produced from the Trametes hirsuta IBB450 strain for the bioremediation of pharmaceutical compounds. Two main laccases mixtures were observed and purified in the extracts and were called Yn and Yg. Peptide fingerprinting analysis suggested that Yn was constituted mainly of laccase Q02497 and Yg of laccase A0A6M5CX58, respectively. Robustness tests, based on tolerance and stability, showed that both laccases were affected in a relatively similar way by salts (KCl, NaCl), organic solvents (ACN, MeOH), denaturing compounds (urea, trypsin, copper) and were virtually unaffected and stable in wastewater. Determination of kinetic constants (Michaelis (KM), catalytic constant (kcat) and kinetic efficiency (K=kcat/KM)) for the transformation of synthetic hormone 17α-ethynylestradiol and the anti-inflammatory agent diclofenac indicates a lower KM and kcat for laccase Yn but relative similar K constant compared to Yg. Synergistic effects were observed for the transformation of diclofenac, unlike 17α-ethynylestradiol. Transformation studies of 17α-ethynylestradiol at different temperatures (4 and 21 °C) indicate a transformation rate reduction of approximately 75-80% at 4 °C against 25% for diclofenac in less than an hour. Finally, the classification of laccases Yg and Yn into one of eight groups (group A-H) suggests that laccase Yg belongs to group A (constitutive laccase) and laccase Yn belongs to group B (inducible laccase).

Is nontargeted data acquisition for target analysis (nDATA) in mass spectrometry a forward-thinking analytical approach?

Targeted mass spectrometry is extensively used for the quantitative measurement of various molecules present in complex matrices. It is certainly one of the most important analytical duties in a mass spectrometry laboratory. Systematic development of selected-reaction monitoring (SRM), multiple-reaction monitoring (MRM) and parallel-reaction monitoring (PRM) methods for targeted mass spectrometry-based analysis was performed without considering future opportunities. The advancement of hardware and software technologies has resulted in greater resolution, accuracy, speed and depth. For sure, SRM, MRM or PRM acquisitions can quantify molecules very accurately at trace levels. However, they do not provide datasets allowing future data mining. Obviously, we cannot truly quantify something that we do not know is there. However, using non-targeted data acquisition for target analysis, we can generate a MS1 and MS2 digital libraries of each sample, providing future proof datasets. This is instrumental for data mining following new questions potentially arising in time permitting new and deeper processing and interpretation. This perspective article provides thoughts on why we believe it is time to question the status quo in targeted mass spectrometry.

Screening of polymer types and chemical weathering in macro- and meso-plastics found on lake and river beaches using a combined chemometric approach.

In the environment, synthetic polymers, commonly known as "plastics", are well-known to undergo various chemical weathering processes, which modify their surface chemistry by introducing new functional groups. Such changes are important to monitor, as they can severely influence the toxicity caused by plastic debris. Therefore, in this study, two chemometric models are proposed to accelerate the chemical classification of macro- and meso-plastics found in the environment. For this purpose, principal component analysis (PCA) and hierarchical cluster analysis (HCA) were applied on preprocessed infrared spectra of 83 plastic fragments found on public lake and river beaches. HCA associated all beach samples with a known plastic, whereas PCA enabled the association of only 39.8% (33 out of 83) of the beach samples with a known plastic. However, both techniques agreed on 93.9% of the samples identified. According to PCA and HCA results, polypropylene and polyethylene were the most frequently identified polymers in the samples. PCA turned out to be a very promising tool for fast screening of weathered plastics, since the distance of samples from the polypropylene cluster in the PCA plot was correlated with weathering. This was later confirmed by employing other characterization techniques such as micro-Raman, X-ray photoelectron spectroscopy and scanning electron microscopy. Finally, future experiments should focus on the applicability of the proposed combined chemometric approach for very small microplastics (<100 µm), as they have more important effects than larger plastics on aquatic ecosystems.

Ultrafast analysis of peptides by laser diode thermal desorption-triple quadrupole mass spectrometry.

RATIONALE: The COVID-19 pandemic demonstrated the importance of high-throughput analysis for public health. Given the importance of surface viral proteins for interactions with healthy tissue, they are targets of interest for mass spectrometry-based analysis. For that reason, the possibility of detecting and quantifying peptides using a high-throughput technique, laser diode thermal desorption-triple quadrupole mass spectrometry (LDTD-QqQMS), was explored. METHODS: Two peptides used as models for small peptides (leu-enkephalin and endomorphin-2) and four tryptic peptides (GVYYPDK, NIDGYFK, IADYNYK, and QIAPGQTGK) specific to the SARS-CoV-2 Spike protein were employed. Target peptides were analyzed individually in the positive mode by LDTD-QqQMS. Peptides were quantified by internal calibration using selected reaction monitoring transitions in pure solvents and in samples spiked with 20 µg mL-1 of a bovine serum albumin tryptic digest to represent real analysis conditions. RESULTS: Low-energy fragment ions (b and y ions) as well as high-energy fragment ions (c and x ions) and some of their corresponding water or ammonia losses were detected in the full mass spectra. Only for the smallest peptides, leu-enkephalin and endomorphin-2, were [M + H]+ ions observed. Product ion spectra confirmed that, with the experimental conditions used in the present study, LDTD transfers a considerable amount of energy to the target peptides. Quantitative analysis showed that it was possible to quantify peptides using LDTD-QqQMS with acceptable calibration curve linearity (R2 > 0.99), precision (RSD < 18.2%), and trueness (bias < 8.3%). CONCLUSIONS: This study demonstrated for the first time that linear peptides can be qualitatively and quantitatively analyzed using LDTD-QqQMS. Limits of quantification and dynamic ranges are still inadequate for clinical applications, but other applications where higher levels of proteins must be detected could be possible with LDTD. Given the high-throughput capabilities of LDTD-QqQMS (>15 000 samples in less than 43 h), more studies are needed to improve the sensitivity for peptide analysis of this technique.

Identifying congeners and transformation products of organic contaminants within complex chemical mixtures in impacted surface waters with a top-down non-targeted screening workflow.

Over 350,000 compounds are registered for production and use including a high number of congeners found in complex chemical mixtures (CCMs). With such a high number of chemicals being released in the environment and degraded into transformation products (TPs), the challenge of identifying contaminants by non-targeted screening (NTS) is massive. "Bottom-up" studies, where compounds are subjected to conditions simulating environmental degradation to identify new TPs, are time consuming and cannot be relied upon to study the TPs of hundreds of thousands of compounds. Therefore, the development of "top-down" workflows, where the structural elucidation of unknown compounds is carried directly on the sample, is of interest. In this study, a top-down NTS workflow was developed using molecular networking and clustering (MNC). A total of 438 compounds were identified including 176 congeners of consumer product additives and 106 TPs. Reference standards were used to confirm the identification of 53 contaminants among them lesser-known pharmaceuticals (aliskiren, sitagliptin) and consumer product additives (lauramidopropyl betaine, 2,2,4-trimethyl-1,2-dihydroquinoline). The MNC tools allowed to group similar TPs and congeners together. As such, several previously unknown TPs of pesticides (metolachlor) and pharmaceuticals (gliclazide, irbesartan) were identified as tentative candidates or probable structures. Moreover, some congeners that had no entry on global repositories (PubChem, ChemSpider) were identified as probable structures. The workflow worked efficiently with oligomers containing ethylene oxide moieties, and with TPs structurally related to their parent compounds. The top-down approach shown in this study addresses several issues with the identification of congeners of industrial compounds from CCMs. Furthermore, it allows elucidating the structure of TPs directly from samples without relying on bottom-up studies under conditions discussed herein. The top-down workflow and the MNC tools show great potential for data mining and retrospective analysis of previous NTS studies.

Uncovering transformation products of four organic contaminants of concern by photodegradation experiments and analysis of real samples from a local river.

In this study, photodegradation experiments simulating the exposure conditions of sunlight on the commonly detected in surface and wastewater contaminants atorvastatin (ATV), bezafibrate (BEZ), oxybenzone (OXZ), and tris(2-butoxyethyl)phosphate (TBEP) were conducted as the fate of these compounds and their transformation products (TPs) was followed. Then a nontargeted analysis was carried out on an urban river to confirm the environmental occurrence of the TPs after which the ECOSAR software was used to generate predicted effect levels of toxicity of the detected TPs on aquatic organisms. Five TPs of ATV were tentatively identified including two stable ones at the end of the experiment: ATV_TP557a and ATV_TP575, that were the product of hydroxylation. Complete degradation of OXZ was observed in the experiment with no significant TP identified. BEZ remained stable and largely undegraded at the end of the exposure. Five TPs of TBEP were found including four that were stable at the end of the experiment: TBEP_TP413, TBEP_TP415, TBEP_TP429, and TBEP_TP343. In the nontargeted analysis, ATV_TP557b, a positional isomer of ATV_TP557a, ATV_TP575 and the 5 TPs of TBEP were tentatively identified. The predicted concentration for effect levels were lower for ATV_TP557b compared to ATV indicating the TP is potentially more toxic than the parent compound. All the TPs of TBEP showed lower predicted toxicity toward aquatic organisms than their parent compound. These results highlight the importance of conducting complete workflows from laboratory experiments, followed by nontargeted analysis to confirm environmental occurrence to end with predicted toxicity to better communicate concern of the newfound TPs to monitoring programs.

Determination of short-chain carboxylic acids and non-targeted analysis of water samples treated by wet air oxidation using gas chromatography-mass spectrometry.

A method based on gas chromatography coupled with electron ionization mass spectrometry employing N,O-bis(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane as derivatization agent was developed to quantify short-chain carboxylic acids (C1-C6) in hospital wastewater treated by wet air oxidation, an advanced oxidation process. Extraction from water and derivatization of volatile and semi-volatile short chain carboxylic acids were optimized and validated and limits of quantification (LOQ = 0.049 mg L-1-4.15 mg L-1), repeatability (RSD = 1.7-12.8%), recovery (31-119%) and trueness (relative bias = -19.0-3.4%) were acceptable. The validated method was successfully applied to monitor the concentration of organic acids formed after wet air oxidation of water samples. Results showed that the method described herein allowed to identify 38% and up to 46% of the final chemical oxygen demand's composition after wet air oxidation of acetaminophen spiked in deionised water and hospital wastewater samples, respectively. The developed method also allowed to perform qualitative non-targeted analysis in hospital wastewater samples after treatment. Results demonstrated that glycerol, methenamine, and benzoic acid were also present in the samples and their presence was confirmed with reference standards.

Non-targeted screening of trace organic contaminants in surface waters by a multi-tool approach based on combinatorial analysis of tandem mass spectra and open access databases.

Non-targeted screening (NTS) in mass spectrometry (MS) helps alleviate the shortcoming of targeted analysis such as missing the presence of concerning compounds that are not monitored and its lack of retrospective analysis to subsequently look for new contaminants. Most NTS workflows include high resolution tandem mass spectrometry (HRMS2) and structure annotation with libraries which are still limited. However, in silico combinatorial fragmentation tools that simulate MS2 spectra are available to help close the gap of missing compounds in empirical libraries. Three NTS tools were combined and used to detect and identify unknown contaminants at ultra-trace levels in surface waters in real samples in this qualitative study. Two of them were based on combinatorial fragmentation databases, MetFrag and the Similar Partition Searching algorithm (SPS), and the third, the Global Natural Products Social Networking (GNPS), was an ensemble of empirical databases. The three NTS tools were applied to the analysis of real samples from a local river. A total of 253 contaminants were identified by combining all three tools: 209 were assigned a probable structure and 44 were confirmed using reference standards. The two major classes of contaminants observed were pharmaceuticals and consumer product additives. Among the confirmed compounds, octylphenol ethoxylates, denatonium, irbesartan and telmisartan are reported for the first time in surface waters in Canada. The workflow presented in this work uses three highly complementary NTS tools and it is a powerful approach to help identify and strategically select contaminants and their transformation products for subsequent targeted analysis and uncover new trends in surface water contamination.

Further studies on the signal enhancement effect in laser diode thermal desorption-triple quadrupole mass spectrometry using microwell surface coatings.

The laser diode thermal desorption (LDTD) ionization source allows ultrafast and sensitive analysis of small molecules by mass spectrometry. Signal enhancement in LDTD has been observed when coating the surface of sample microwells with a solution of ethylenediaminetetraacetic acid (EDTA) or nitrilotriacetic acid. Here we present a quantitative analysis of signal enhancement using solutions of diverse commercial proteins (lysozyme, immunoglobulin G, albumin, and fibrinogen) as coatings. Results showed that compounds with polar chemical functions such as carboxylic acid, sulfonyl, and nitro had signal enhancement factors, in most cases higher than 10, when using any of the tested proteins as coating agent. Analysis of variance revealed that immunoglobulin G and fibrinogen gave the best results. However, the signal enhancement factors obtained with these proteins were not superior to those observed with EDTA. To explain the signal enhancement effect of proteins, analysis by scanning electron microscopy of dried samples on the microwell sample plates was carried out. Images showed that salicylic acid, one of the compounds with the highest observed signal enhancement, formed a thick layer when applied directly on the uncoated surface, but it formed small crystals (<1 µm) in the presence of protein or EDTA coatings. Further crystallographic studies using powder X-ray diffraction showed that the crystalline form of salicylic acid is modified in the presence of EDTA. Salicylic acid when mixed with EDTA had a higher percentage of amorphous phase (38.1%) than without EDTA (23.1%). These results appear to confirm that the diminution of crystal size of analytes and the increase of amorphous phase are implicated in signal enhancement effect observed in LDTD using microwell surface coatings. To design better coatings and completely elucidate the signal enhancement effect in LDTD, more studies are necessary to understand the effects of coatings on the ionization of analytes.

The NSERC Canadian Lake Pulse Network: A national assessment of lake health providing science for water management in a changing climate.

The distribution and quality of water resources vary dramatically across Canada, and human impacts such as land-use and climate changes are exacerbating uncertainties in water supply and security. At the national level, Canada has no enforceable standards for safe drinking water and no comprehensive water-monitoring program to provide detailed, timely reporting on the state of water resources. To provide Canada's first national assessment of lake health, the NSERC Canadian Lake Pulse Network was launched in 2016 as an academic-government research partnership. LakePulse uses traditional approaches for limnological monitoring as well as state-of-the-art methods in the fields of genomics, emerging contaminants, greenhouse gases, invasive pathogens, paleolimnology, spatial modelling, statistical analysis, and remote sensing. A coordinated sampling program of about 680 lakes together with historical archives and a geomatics analysis of over 80,000 lake watersheds are used to examine the extent to which lakes are being altered now and in the future, and how this impacts aquatic ecosystem services of societal importance. Herein we review the network context, objectives and methods.

Signal enhancement in laser diode thermal desorption-triple quadrupole mass spectrometry analysis using microwell surface coatings.

Laser-diode thermal desorption (LDTD) is an ionization source usually coupled to triple quadrupole mass spectrometry (QqQMS) and specifically designed for laboratories requiring high-throughput analysis. It has been observed that surface coatings on LDTD microwell plates can improve the sensitivity of the analysis of small polar molecules. The objective of the present study is to understand and quantify the effect of microwell surface coatings on signal intensity of small organic molecules of clinical, environmental, and forensic interest. Experiments showed that the peak areas of diclofenac, chloramphenicol, salicylic acid, and 11-nor-9-carboxy-Δ9 -tetrahydrocannabinol obtained by LDTD-QqQMS increased by up to 3 orders of magnitude when using microwells coated with ethylenediaminetetraacetic acid (EDTA). Tests with different chelating agents and polytetrafluoroethylene as microwell surface coatings showed that nitrilotriacetic acid gave significantly higher peak areas for five out of the nine compounds that showed signal enhancement using chelating agents as coatings. Scanning electron microscopy studies of EDTA-coated and uncoated microwells showed that analytes deposited in the former formed more uniform and thinner films than in the latter. The enhancement effect of surface coatings in LDTD-QqQMS was explained mainly by the formation of homogenous and thinner layers of nanocrystals of analytes that are easier to desorb thermally than the layers formed when the analytes dry in direct contact with the bare stainless-steel surface. Chemisorption of some analytes to the stainless-steel surface of the microwell plate appeared to be a minor factor. Surface coatings widen the number of compounds analyzable by LDTD-QqQMS and can also improve sensitivity and limits of detection.

Identifying Fenton-Reacted Trimethoprim Transformation Products Using Differential Mobility Spectrometry.

A transformation product of trimethoprim, a contaminant of emerging concern in the environment, is generated using an electro-assisted Fenton reaction and analyzed using differential mobility spectrometry (DMS) in combination with MS/MS techniques and quantum chemical calculations to develop a rapid method for identification. DMS is used as a prefilter to separate positional isomers prior to subsequent identification by mass spectrometric analyses. Collision induced dissociation of each DMS separated species is used to reveal fragmentation patterns that can be correlated to specific isomer structures. Analysis of the experimental data and supporting quantum chemical calculations show that methylene-hydroxylated and methoxy-containing phenyl ring hydroxylated transformation products are observed. The proposed methodology outlines a high-throughput technique to determine transformation products of small molecules accurately, in a short time and requiring minimal sample concentrations (<25 ng/mL).

Application of Spectral Accuracy to Improve the Identification of Organic Compounds in Environmental Analysis.

Correct identification of a chemical substance in environmental samples based only on accurate mass measurements can be difficult especially for molecules >300 Da. Here is presented the application of spectral accuracy, a tool for the comparison of isotope patterns toward molecular formula generation, as a complementary technique to assist in the identification process of organic micropollutants and their transformation products in surface water. A set of nine common contaminants (five antibiotics, an herbicide, a beta-blocker, an antidepressant, and an antineoplastic) frequently found in surface water were spiked in methanol and surface water extracts at two different concentrations (80 and 300 µg L-1). They were then injected into three different mass analyzers (triple quadrupole, quadrupole-time-of-fight, and quadrupole-orbitrap) to study the impact of matrix composition, analyte concentration, and mass resolution on the correct identification of molecular formulas using spectral accuracy. High spectral accuracy and ranking of the correct molecular formula were in many cases compound-specific due principally to conditions affecting signal intensity such as matrix effects and concentration. However, in general, results showed that higher concentrations and higher resolutions favored ranking the correct formula in the top 10. Using spectral accuracy and mass accuracy it was possible to reduce the number of possible molecular formulas for organic compounds of relative high molecular mass (e.g., between 400 and 900 Da) to less than 10 and in some cases, it was possible to unambiguously assign one specific molecular formula to an experimental isotopic pattern. This study confirmed that spectral accuracy can be used as a complementary diagnostic technique to improve confidence levels for the identification of organic contaminants under environmental conditions.

Quantification of ecdysteroids and retinoic acids in whole daphnids by liquid chromatography-triple quadrupole mass spectrometry.

Quantification of ecdysteroids and retinoic acids at picograms per individual is typically achieved with radioimmunoassay methods. However, those methods cannot identify individual types of ecdysteroids or provide an absolute concentration, which poses problems for comparative assays such as the metabolic profiling approach for toxicity testing. The method described in the present paper, based on liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry, was developed to allow the quantification in whole daphnids extracts of ecdysteroids (20-hydroxyecdysone, ecdysone, ponasterone A) and retinoic acid (sum of isomers). This approach avoids having to perform the difficult task of sampling the haemolymph on small organism (<5mm). Recoveries, evaluated at three concentrations in matrix blank fortified samples, ranged from 83 to 119% for ecdysteroids and from 144 to 155% for retinoic acids. Precision (2.4-14.2%) and accuracy (-41.7 to 14.5%) were reproducible and stable over three quality control concentrations. The described liquid chromatography-triple quadrupole mass spectrometry method achieved quantification limits ranging from 210 to 380 pg mL(-1) for ecdysteroids and 5 ng mL(-1) for retinoic acids in spiked matrix blanks. 20-hydroxyecdysone was quantified in Daphnia magna adults (19 ± 8 pg ind(-1)) and juveniles (3.6 ± 1.0 pg ind(-1)), but was below the limit of quantification in neonates (≈ 0.19 pg ind(-1)). Ecdysone was also detected in adult specimens (≈ 1.8 pg ind(-1)).

Linking drugs of abuse in wastewater to contamination of surface and drinking water.

The concentrations of 17 drugs of abuse, including cocaine, several amphetamines, opioid drugs, and 2 metabolites--benzoylecgonine, a metabolite of cocaine, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrolidine, a metabolite of methadone--were investigated in an urban watershed that is heavily impacted by discharges of municipal wastewater. The artificial sweetener sucralose was also monitored as a persistent tracer of contamination from municipal wastewater. Monitoring was conducted in a municipal wastewater treatment plant (WWTP) and at sites upstream and downstream of the WWTP discharge, as well as in a drinking water treatment plant (DWTP) located 19 km downstream of the WWTP discharge that withdraws raw water from the river. Drug concentrations were monitored with polar organic chemical integrative samplers deployed for 2 wk in the river and in the WWTP and DWTP. Several of the investigated compounds exhibited a decrease in concentration with distance downstream from the wastewater discharge into the river, but there was little attenuation of sucralose, cocaine, benzoylecgonine, morphine, acetylmorphine, acetylcodeine, and oxycodone. Heroin and methadone were not detected at any sample locations. Amphetamine, methamphetamine, 3,4-methylenedioxy-methamphetamine, and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrolidine were not detected in the samples collected at the drinking water intake. Many of the drugs of abuse were not removed effectively in the DWTP, including cocaine, benzoylecgonine, methylenedioxyamphetamine, ephedrine, and several prescription opioids, most probably because the DWTP was operating at or above its rated treatment capacity. These data indicate that there can be transport of drugs of abuse from wastewater sources into drinking water in urban watersheds.

Application of XCMS Online and toxicity bioassays to the study of transformation products of levofloxacin.

We studied the nature and antimicrobial activity of ozonolysis transformation products (OTPs) of levofloxacin (LEV), a frequently detected fluoroquinolone antimicrobial in environmental waters. Two bioassays, the Kirby-Bauer test and the broth microdilution assay, were used to measure changes in the antimicrobial activity of solutions at low LEV to O3 molar ratios (2:1, 2:3 and 1:3) compared to solutions without added O3 (LEV:O3 1:0). The Kirby-Bauer test was not sensitive enough to detect significant differences in the growth inhibition zones in samples LEV:O3 2:1 and LEV:O3 1:0; however, the broth microdilution assay showed that bacterial growth inhibition was significantly lower (P<0.001) in the solutions exposed to O3. Loss of antimicrobial activity in LEV:O3 2:1 solutions of (48±16)% was in agreement with the concentration decrease of LEV of (36±3)% in those same samples. A method of identification of OTPs using XCMS Online was applied to LEV:O3 2:1 and 1:0 samples and indicated the presence of an OTP of LEV of formula C18H20O5N3F, which was identified as LEV-N-oxide. The molecular structure of this compound was partially confirmed by tandem mass spectrometry experiments. This study showed that even at sub-optimal ozone doses, OTPs of higher antimicrobial activity than LEV were not formed.