De facto Water Reuse: Investigating the Fate and Transport of Chemicals of Emerging Concern from Wastewater Discharge through Drinking Water Treatment Using Non-targeted Analysis and Suspect Screening.

Wastewater is a source for many contaminants of emerging concern (CECs), and surface waters receiving wastewater discharge often serve as source water for downstream drinking water treatment plants. Nontargeted analysis and suspect screening methods were used to characterize chemicals in residence-time-weighted grab samples and companion polar organic chemical integrative samplers (POCIS) collected on three separate hydrologic sampling events along a surface water flow path representative of de facto water reuse. The goal of this work was to examine the fate of CECs along the study flow path as water is transported from wastewater effluent through drinking water treatment. Grab and POCIS samples provided a comparison between residence-time-weighted single-point and integrative sample results. This unique and rigorous study design, coupled with advanced analytical chemistry tools, provided important insights into chemicals found in drinking water and their potential sources, which can be used to help prioritize chemicals for further study. K-means clustering analysis was used to identify patterns in chemical occurrences across both sampling sites and sampling events. Chemical features that occurred frequently or survived drinking water treatment were prioritized for identification, resulting in the probable identification of over 100 CECs in the watershed and 28 CECs in treated drinking water.

Enhanced efficiency of MS/MS all-ion fragmentation for non-targeted analysis of trace contaminants in surface water using multivariate curve resolution and data fusion.

Data-independent acquisition-all-ion fragmentation (DIA-AIF) mode of mass spectrometry can facilitate wide-scope non-target analysis of contaminants in surface water due to comprehensive spectral identification. However, because of the complexity of the resulting MS2 AIF spectra, identifying unknown pollutants remains a significant challenge, with a significant bottleneck in translating non-targeted chemical signatures into environmental impacts. The present study proposes to process fused MS1 and MS2 data sets obtained from LC-HRMS/MS measurements in non-targeted AIF workflows on surface water samples using multivariate curve resolution-alternating least squares (MCR-ALS). This enables straightforward assignment between precursor ions obtained from resolved MS1 spectra and their corresponding MS2 spectra. The method was evaluated for two sets of tap water and surface water contaminated with 14 target chemicals as a proof of concept. The data set of surface water samples consisting of 3506 MS1 and 2170 MS2 AIF mass spectral features was reduced to 81 components via a fused MS1-MS2 MCR model that describes at least 98.8% of the data. Each component summarizes the distinct chromatographic elution of components together with their corresponding MS1 and MS2 spectra. MS2 spectral similarity of more than 82% was obtained for most target chemicals. This highlights the potential of this method for unraveling the composition of MS/MS complex data in a water environment. Ultimately, the developed approach was applied to the retrospective non-target analysis of an independent set of surface water samples.

Antiproliferative Effects of Methanolic Fruit Extract of Solanum xenthocarpum (L.) on Human Breast Cancer Cells.

Solanum xanthocarpum, a perennial herb native to India, contains steroidal glycoalkaloids with notable anticancer properties. This study investigated the antioxidant and antiproliferative effects of methanolic fruit extract of S. xanthocarpum on human breast cancer cells (MDA-MB-231). Phytochemical screening and LC-HRMS analysis confirmed presence of various primary and secondary metabolites. Antioxidant activity was assessed through DPPH, ABTS radical scavenging, reducing power, and phosphomolybdate assays. The extract demonstrated significant antioxidant potential with EC50 values of 60.10 ± 0.88 µg/mL (DPPH) and 392.29 ± 3.93 µg/mL (ABTS). Cytotoxicity against MDA-MB-231 cells was evaluated via morphological analysis, MTT assays, and IC50 determination (24.19 ± 0.56 µg/L). Apoptosis was confirmed using dual staining techniques (AO/EB, Hoechst 33342/PI, DAPI), revealing condensed nuclei, apoptotic bodies, and reduced mitochondrial membrane potential, as indicated by Rhodamine staining. Additionally, increased reactive oxygen species (ROS) levels were observed using H2-DCF-DA staining. The total phenolic and flavonoid contents of the extract were 127.78 ± 3.547 mg GAE/g and 98.06 ± 4.289 mg QE/g, respectively. These findings suggest that the methanolic fruit extract of S. xanthocarpum possesses strong antioxidant and anticancer activities, indicating its potential role in cancer treatment. Further studies are warranted to explore its bioactive compounds for developing novel anticancer therapies.

In Vitro and In Vivo Human Metabolism of Ostarine, a Selective Androgen Receptor Modulator and Doping Agent.

Ostarine (enobasarm) is a selective androgen receptor modulator with great therapeutic potential. However, it is also used by athletes to promote muscle growth and enhance performances without the typical adverse effects of anabolic steroids. Ostarine popularity increased in recent years, and it is currently the most abused "other anabolic agent" (subclass S1.2. of the "anabolic agents" class S1) from the World Anti-Doping Agency's (WADA) prohibited list. Several cases of liver toxicity were recently reported in regular users. Detecting ostarine or markers of intake in biological matrices is essential to document ostarine use in doping. Therefore, we sought to investigate ostarine metabolism to identify optimal markers of consumption. The substance was incubated with human hepatocytes, and urine samples from six ostarine-positive cases were screened. Analyses were performed via liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) and software-assisted data mining, with in silico metabolite predictions. Ten metabolites were identified with hydroxylation, ether cleavage, dealkylation, O-glucuronidation, and/or sulfation. The production of cyanophenol-sulfate might participate in the mechanism of ostarine liver toxicity. We suggest ostarine-glucuronide (C25H22O9N3F3, diagnostic fragments at m/z 118, 185, and 269) and hydroxybenzonitrile-ostarine-glucuronide (C25H22O10N3F3, diagnostic fragments at m/z 134, 185, and 269) in non-hydrolyzed urine and ostarine and hydroxybenzonitrile-ostarine (C19H14O4N3F3, diagnostic fragments at m/z 134, 185, and 269) in hydrolyzed urine as markers to document ostarine intake in doping.

Chemical Composition of Methanol Extracts from Leaves and Flowers of Anemonopsis macrophylla (Ranunculaceae).

Anemonopsis Siebold et Zucc. is an unstudied single-species genus belonging to the tribe Cimicifugeae (Ranunculaceae). The only species of this genus-Anemonopsis macrophylla Siebold and Zucc.-is endemic to Japan. There are no data on its chemical composition. This work is the first to determine (with liquid chromatography-high-resolution mass spectrometry, LC-HRMS) the chemical composition of methanol extracts of leaves and flowers of A. macrophylla. More than 100 compounds were identified. In this plant, the classes of substances are coumarins (13 compounds), furocoumarins (3), furochromones (2), phenolic acids (21), flavonoids (27), and fatty acids and their derivatives (15 compounds). Isoferulic acid (detected in extracts from this plant) brings this species closer to plants of the genus Cimicifuga, one of the few genera containing this acid and ferulic acid at the same time. Isoferulic acid is regarded as a reference component of a quality indicator of Cimicifuga raw materials. The determined profiles of substances are identical between the leaf and flower methanol extracts. Differences in levels of some identified substances were revealed between the leaf and flower extracts of A. macrophylla; these differences may have a substantial impact on the manifestation of the biological and pharmacological effects of the extracts in question.

Exploratory Untargeted Metabolomics of Dried Blood Spot Samples from Newborns with Maple Syrup Urine Disease.

Currently, tandem mass spectrometry-based newborn screening (NBS), which examines targeted biomarkers, is the first approach used for the early detection of maple syrup urine disease (MSUD) in newborns, followed by confirmatory genetic mutation tests. However, these diagnostic approaches have limitations, demanding the development of additional tools for the diagnosis/screening of MUSD. Recently, untargeted metabolomics has been used to explore metabolic profiling and discover the potential biomarkers/pathways of inherited metabolic diseases. Thus, we aimed to discover a distinctive metabolic profile and biomarkers/pathways for MSUD newborns using untargeted metabolomics. Herein, untargeted metabolomics was used to analyze dried blood spot (DBS) samples from 22 MSUD and 22 healthy control newborns. Our data identified 210 altered endogenous metabolites in MSUD newborns and new potential MSUD biomarkers, particularly L-alloisoleucine, methionine, and lysoPI. In addition, the most impacted pathways in MSUD newborns were the ascorbate and aldarate pathways and pentose and glucuronate interconversions, suggesting that oxidative and detoxification events may occur in early life. Our approach leads to the identification of new potential biomarkers/pathways that could be used for the early diagnosis/screening of MSUD newborns but require further validation studies. Our untargeted metabolomics findings have undoubtedly added new insights to our understanding of the pathogenicity of MSUD, which helps us select the appropriate early treatments for better health outcomes.

[Application of chromatographic retention time correction in untargeted screening].

OBJECTIVE: Chromatographic retention time correction is one of the important steps to effectively improve the accuracy of identification. This article proposed a strategy for untargeted screening of biological samples based on retention time correction. METHODS: A pre-treatment method for biological samples was established. The conditions of liquid chromatography and mass spectrometry were optimized. Fourteen compounds were selected as calibration agents. The retention time correction of different samples, different injection time, different brands of instruments, changing chromatographic column and changing mobile phase were investigated. RESULTS: Calibration agents had a wide coverage, good stability and no interference with sample determination. They could be uniformly distributed in the chromatogram in both positive and negative ion modes. The chromatogram was divided into several time intervals. Calibration agents in each time period were used for retention time linear correction, and the correction effects were good. CONCLUSION: The retention time correction method could eliminate the retention time drift caused by experimental conditions, improve the accuracy of qualitative analysis, and help to solve the problem of high false positive result based on mass spectrum information.

Contamination Pattern and Risk Assessment of Polar Compounds in Snow Melt: An Integrative Proxy of Road Runoffs.

To assess the contamination and potential risk of snow melt with polar compounds, road and background snow was sampled during a melting event at 23 sites at the city of Leipzig and screened for 489 chemicals using liquid chromatography high-resolution mass spectrometry with target screening. Additionally, six 24 h composite samples were taken from the influent and effluent of the Leipzig wastewater treatment plant (WWTP) during the snow melt event. 207 compounds were at least detected once (concentrations between 0.80 ng/L and 75 µg/L). Consistent patterns of traffic-related compounds dominated the chemical profile (58 compounds in concentrations from 1.3 ng/L to 75 µg/L) and among them were 2-benzothiazole sulfonic acid and 1-cyclohexyl-3-phenylurea from tire wear and denatonium used as a bittern in vehicle fluids. Besides, the analysis unveiled the presence of the rubber additive 6-PPD and its transformation product N-(1.3-dimethylbutyl)-N'-phenyl-p-phenylenediamine quinone (6-PPDQ) at concentrations known to cause acute toxicity in sensitive fish species. The analysis also detected 149 other compounds such as food additives, pharmaceuticals, and pesticides. Several biocides were identified as major risk contributors, with a more site-specific occurrence, to acute toxic risks to algae (five samples) and invertebrates (six samples). Ametryn, flumioxazin, and 1,2-cyclohexane dicarboxylic acid diisononyl ester are the main compounds contributing to toxic risk for algae, while etofenprox and bendiocarb are found as the main contributors for crustacean risk. Correlations between concentrations in the WWTP influent and flow rate allowed us to discriminate compounds with snow melt and urban runoff as major sources from other compounds with other dominant sources. Removal rates in the WWTP showed that some traffic-related compounds were largely eliminated (removal rate higher than 80%) during wastewater treatment and among them was 6-PPDQ, while others persisted in the WWTP.

Detection of synthetic analogues of insulin-like growth factor 1 in different biological fluids by liquid chromatography quadrupole time-of-flight mass spectrometry: comparison of different immunoaffinity protocols.

Insulin-like growth factor 1 analogues are prohibited in sport for their ability to enhance athletic performance in several sport disciplines. Their detection presents several analytical challenges, mainly due to the minimum required performance limits fixed by the World Anti-Doping Agency. Here, we are presenting analytical workflows to detect IGF-1 and its analogues in different biological matrices. Several off-line immunocapture techniques and protocols were comparatively evaluated. Separation and detection were performed by using standard flow reverse-phase liquid chromatography coupled to a time-of-flight mass spectrometer. The best recoveries were obtained using magnetic beads or pipette tips functionalized with protein A. The analytical workflows were fully validated for qualitative determinations: all the target analytes were clearly distinguishable from the interference of the matrices, with limits of detection and identification in the range of 0.05-0.30 ng/mL in urine and 0.5-2.0 ng/mL in serum/plasma. The extraction efficiency proved to be repeatable (CV% < 10) with recoveries higher than 50%. Intra- and inter-day precision were found to be smaller than 10 and 15%, respectively. The method was successfully applied to the analysis of authentic matrix samples containing the target peptides at the minimum required performance limits, proving that the method developed can be successfully applied to detect and identify IGF-1 analogues for doping control purposes in all the matrices selected. The analytical workflow developed here to detect the target peptides in different matrices can be readily implemented in anti-doping laboratories and has the potential to be adapted for the simultaneous analysis of different similarly sized peptide hormones of doping relevance.

In vitro metabolic profiling of new synthetic cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA.

In the recreational drug market, synthetic cannabinoids with a new acetamide linker structure emerged, most likely to circumvent the law. As the knowledge of drug metabolites is vital for proving drug consumption, the phase I metabolism of the newly emerging cannabinoids, ADB-FUBIATA, AFUBIATA, CH-FUBIATA, and CH-PIATA, was investigated. Each drug (10 µmol/L) was incubated with human liver microsomes for 1 h, and the samples, after dilution, were analyzed by liquid chromatography-high-resolution mass spectrometry. All drugs were metabolized via hydroxylation and N-dealkylation, while AFUBIATA and CH-PIATA additionally underwent ketone formation. The metabolites AF7 (hydroxylated at the indole/adjacent methylene) of ADB-FUBIATA, A16 (hydroxylated at the adamantane) of AFUBIATA, CF15 (hydroxylated at the cyclohexane) of CH-FUBIATA, and CP9 (hydroxylated at the pentane) of CH-PIATA were the most abundant metabolites by considering the peak areas on the chromatograms, and are recommended for urinalysis. The structure-metabolism relationship was also discussed, which generally agreed well with previously reported metabolic pathways of other synthetic cannabinoids. However, the preferred hydroxylation site of ADB-FUBIATA, the indole/adjacent methylene, clearly differed from that of ADB-FUBICA, the 3,3-dimethylbutanamide moiety, despite their structures differing only by a methylene group, emphasizing that metabolic predictions of new drugs should not replace in vitro experimental analyses, albeit helpful.