Development of a robust non-targeted analysis approach for fast identification of endocrine disruptors and their metabolites in human urine for exposure assessment.

Endocrine disrupting chemicals are of concern because of possible human health effects, thus they are frequently included in biomonitoring studies. Current analytical methods are focused on known chemicals and are incapable of identifying or quantifying other unknown chemicals and their metabolites. Non-targeted analysis (NTA) methods are advantageous since they allow for broad chemical screening, which provides a more comprehensive characterization of human chemical exposure, and can allow elucidation of metabolic pathways for unknown chemicals. There are still many challenges associated with NTA, which can impact the results obtained. The chemical space, i.e., the group of known and possible compounds within the scope of the method, must clearly be defined based on the sample preparation, as this is critical in identifying chemicals with confidence. Data acquisition modes and mobile phase additives used with liquid chromatography coupled to high-resolution mass-spectrometry can affect the chemicals ionized and structural identification based on the spectral quality. In this study, a sample preparation method was developed using a novel clean-up approach with CarbonS cartridges, for endocrine-disrupting chemicals in urine, including new bisphenol A analogues and benzophenone-based UV filters, like methyl bis (4-hydroxyphenyl acetate). The study showed that data dependent acquisition (DDA) had a lower identification rate (40%) at low spiking levels, i.e., 1 ng/mL, compared to data independent acquisition (DIA) (57%), when Compound Discoverer was used. In DDA, more compounds were identified using Compound Discoverer, with an identification rate of 95% when ammonium acetate was compared to acetic acid (82%) as a mobile phase additive. TraceFinder software had an identification rate of 53% at 1 ng/mL spiking level using the DDA data, compared to 40% using the DIA data. Using the developed method, 2,4 bisphenol F was identified for the first time in urine samples. The results show how NTA can provide human exposure information for risk assessment and regulatory action but standardized reporting of procedures is needed to ensure study results are reproducible and accurate.

Influence of data acquisition modes and data analysis approaches on non-targeted analysis of phthalate metabolites in human urine.

Humans are often exposed to phthalates and their alternatives, on account of their widespread use in PVC as plasticizers, which are associated with harmful human effects. While targeted biomonitoring provides quantitative information for exposure assessment, only a small portion of phthalate metabolites has been targeted. This results in a knowledge gap in human exposure to other unknown phthalate compounds and their metabolites. Although the non-targeted analysis (NTA) approach is capable of screening a broad spectrum of chemicals, there is a lack of harmonized workflow in NTA to generate reproducible data within and between different laboratories. The objective of this study was to compare two different NTA data acquisition modes, the data-dependent (DDA) and independent (DIA) acquisition (DDA), as well as two data analysis approaches, based on diagnostic ions and Compound Discoverer software for the prioritization of candidate precursors and identification of unknown compounds in human urine. Liquid chromatography coupled to high-resolution mass spectrometry was used for sample analysis. The combination of three-diagnostic-ion extraction and DDA data acquisition was able to improve data filtering and data analysis for prioritizing phthalate metabolites. With DIA, 25 molecular features were identified in human urine, while 32 molecular features were identified in the same urine samples using DDA data. The number of molecular features identified with level 1 confidence was 11 and 9 using DIA and DDA data, respectively. The study demonstrated that besides sample preparation, the impact of data acquisition must be taken into account when developing a NTA method and a consistent protocol for evaluating such an impact is necessary.

Fish as sentinels of antimicrobial resistant bacteria, epidemic carbapenemase genes, and antibiotics in surface water.

Surface waters, especially those receiving wastewater flows, can disseminate antimicrobial resistant bacteria (ARB), antimicrobial resistance genes (ARG), and antibiotics. In the Scioto River of central Ohio, United States, we evaluated fishes as potential sentinels of ARB and antimicrobial contamination and investigated the influence of antimicrobial exposure on the fish intestinal resistome. Seventy-seven fish were collected from river reaches receiving inputs from two wastewater treatment plants that serve the greater Columbus Metropolitan Area. Fish were screened for the presence of cephalosporin-resistant (CeRO) and carbapenem-resistant (CRO) organisms, epidemic carbapenemase genes, and antibiotic drugs and metabolites using culture methods, droplet digital PCR, and ultra-high performance liquid chromatography tandem mass spectroscopy (UHPLC-MS/MS). Nearly 21% of fish harbored a CeRO in their resistome, with 19.4% exhibiting bacteria expressing an AmpC genotype encoded by blaCMY, and 7.7% with bacteria expressing an extended-spectrum ß-lactamase phenotype encoded by blaCTX-M. blaKPC and blaNDM were present in 87.7% (57/65) and 80.4% (37/46) of the intestinal samples at an average abundance of 104 copies. Three antibiotics-lincomycin (19.5%), azithromycin (31.2%) and sulfamethoxazole (3.9%)-were found in hepatic samples at average concentrations between 25-31 ng/g. Fish harboring blaCTX-M and those exposed to azithromycin were at greater odds of being downstream of a wastewater treatment plant. Fish that bioconcentrated antibiotics in their liver were not at greater odds of harboring CeRO, CRO, or epidemic carbapenemase gene copies in their resistome. Our findings confirm that fishes can be effective bioindicators of surface waters contaminated with ARB, ARG, and antibiotics. Moreover, our findings highlight the varying importance of different mechanisms that facilitate establishment of ARB in aquatic ecosystems.

Application of Nontarget Analysis and High-Resolution Mass Spectrometry for the Identification of Thermal Transformation Products of Oxytetracycline in Pacific White Shrimp.

ABSTRACT: Oxytetracycline (OTC) is an antibiotic authorized for use in aquaculture; it is often detected in seafood products, especially shrimp. Previous studies investigating the fate of OTC in shrimp tissues after cooking were limited to quantification of parent compound residues and did not describe any potential transformation products formed. Hence, the main objective of this study was to apply a nontarget analysis workflow to study the fate of OTC in shrimp muscle. Furthermore, "water" and "spiked" models were evaluated for their suitability to track the transformation of OTC in incurred muscle and to determine whether the matrix plays a role in the transformation pathway. First, four different extraction methods were compared for the determination of OTC in muscle. Second, raw and cooked samples were then extracted using a suitable method (acidified water-methanol-acetonitrile, with cleanup of samples achieved using freezing) and were analyzed by high-performance liquid chromatography quadrupole time-of-flight mass spectrometry. OTC levels were reduced by 75 and 87% in muscle and water, respectively. Identification of thermal transformation products was limited to formula generation, but results showed that different compounds were identified in spiked and incurred muscle.

Evaluation of different extractions for the metabolite identification of malachite green in brook trout and shrimp.

Applications of mass spectrometry-based metabolomics in food science have developed fast in the last decade. Sample preparation and data processing are critical in non-target/metabolomic workflows but there is currently no standardized protocol for the development of these methods. The impact of data processing parameters or the inclusion of a different matrix is not often taken into account during the selection of an extraction. Thus, this study aimed to investigate the impact of different extractions, e.g., QuEChERS, and data processing on the determination of malachite green metabolites in two different organisms, brook trout and shrimp. The results obtained confirm the need for a harmonized approach for the validation of non-target workflows, as depending on the comparison criteria, the matrix, the mode of ionization or data processing, a different extraction could be chosen. This study also identified for the first time des-methylated leucomalachite green as another metabolite in the two organisms.

Application of non-target analysis to study the thermal transformation of malachite and leucomalachite green in brook trout and shrimp.

The fate of malachite green and its main metabolite leucomalachite green during thermal treatment was examined in seafood (brook trout and white shrimp) using non-target analysis. Samples were extracted using QuEChERS and analyzed using liquid chromatography coupled with quadruple time of flight mass spectrometry. Malachite green levels were reduced in meat during boiling (∼40%), microwaving (64%), and canning (96%). Only microwaving was successful in significantly decreasing leucomalachite green levels in brook trout. The reduction percentages of the two target analytes were not significantly different in shrimp (mean fat content = 0.8 ± 0.3%) and in brook trout (mean fat content = 3.5 ± 1.7%), suggesting that a higher fat content may not affect the reduction of the more lipophilic leucomalachite green in these two matrices. Three transformation products were tentatively identified in the cooked tissues, resulting from the cleavage of the conjugated structure or through demethylation. Further research is needed to determine possible adverse health effects. The findings of this study show how non-target analysis can complement targeted methodologies in identifying and evaluating risks to human health.

Suspect screening of pharmaceuticals in fish livers based on QuEChERS extraction coupled with high resolution mass spectrometry.

The presence of pharmaceuticals and personal care products (PPCPs) in aquatic environments is of increasing concern due to the presence of residues in fish and aquatic organisms, and emerging antibiotic resistance. Wastewater release is an important contributor to the presence of these compounds in aquatic ecosystems, where they may accumulate in food webs. The traditional environmental surveillance approach relies on the targeted analysis of specific compounds, but more suspect screening methods have been developed recently to allow for the identification of a variety of contaminants. In this study, a method based on QuEChERS extraction - using acetonitrile/water mixture as solvent and PSA/C18 for sample clean-up - was applied to identify pharmaceuticals and their metabolites in fish livers. Both target and suspect screening workflows were used and fish were sampled upstream and downstream of wastewater treatment plants from the Scioto River, Ohio (USA). The method performed well in terms of extraction of some target PPCPs, with recoveries generally above 90%, good repeatability (<20%), and linearity. Based on target analysis, lincomycin and sulfamethoxazole were two antibiotics identified in fish livers at average concentrations of 30.3 and 25.6 ng g-1 fresh weight, respectively. Using suspect screening, another antibiotic, azithromycin and an antidepressant metabolite, erythrohydrobupropion were identified (average concentrations: 27.8 and 13.8 ng g-1, respectively). The latter, reported, to the best of our knowledge, for the first time in fish livers, was also found at higher concentrations in fish livers sampled downstream vs. upstream. The higher frequency of detection for azithromycin in benthic feeding fish species (63%) as well as clusters identified between different foraging groups suggest that foraging behavior may be an important mechanism in the bioaccumulation of PPCPs. This study shows how suspect screening is effective in identifying new contaminants in fish livers, notably using differential analysis among different spatially distributed samples.