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.

Natural toxins produced by Alternaria fungi include the mycotoxins alternariol, tenuazonic acid and altertoxins I and II. Several of these toxins have shown high toxicity even at low levels including genotoxic, mutagenic, and estrogenic effects. However, the metabolic effects of toxin exposure from Alternaria are understudied, especially in the liver as a key target. To gain insight into the impact of Alternaria toxin exposure on the liver metabolome, rats (n = 21) were exposed to either (1) a complex culture extract with defined toxin profiles from Alternaria alternata (50 mg/kg body weight), (2) the isolated, highly genotoxic altertoxin-II (ATX-II) (0.7 mg/kg of body weight) or (3) a solvent control. The complex mixture contained a spectrum of Alternaria toxins including a controlled dose of ATX-II, matching the concentration of the isolated ATX-II. Liver samples were collected after 24 h and analyzed via liquid chromatography-high-resolution mass spectrometry (LC-HRMS). Authentic reference standards (> 100) were used to identify endogenous metabolites and exogenous compounds from the administered exposures in tandem with SWATH-acquired MS/MS data which was used for non-targeted analysis/screening. Screening for metabolites produced by Alternaria revealed several compounds solely isolated in the liver of rats exposed to the complex culture, confirming results from a previously performed targeted biomonitoring study. This included the altersetin and altercrasin A that were tentatively identified. An untargeted metabolomics analysis found upregulation of acylcarnitines in rats receiving the complex Alternaria extract as well as downregulation of riboflavin in rats exposed to both ATX-II and the complex mixture. Taken together, this work provides a mechanistic view of Alternari toxin exposure and new suspect screening insights into hardly characterized Alternaria toxins.

Two protocols of protein extraction from Arthrospira platensis (spirulina) microalgae to study their proteome by mass spectrometry (MS) are here presented. The first is based on an aqueous buffer solution of Tris-HCl and the second on cold acetone. The identification of proteins was carried out by a bottom-up approach, which involves enzymatic digestion of extracted proteins followed by either matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) MS or liquid chromatography (LC) coupled with electrospray ionization (ESI) and Fourier-transform tandem MS. While MALDI-TOF MS allowed for a fast peptide mass fingerprinting (PMF) check yet identifying less than 20 proteins in the extracted samples, the data-dependent acquisitions (DDA) mode of reversed-phase (RP) LC-ESI tandem FTMS/MS separations allowed us to recognize more than one hundred proteins by searching into dedicated spectral libraries. The application of MALDI-TOF MS analysis was found, however, of great support for preliminary investigations of cyanobacteria samples before proceeding with the RPLC-ESI-MS/MS DDA investigation, which definitively allows for a qualitative proteome analysis also of minor spirulina proteins in processed foodstuffs. Although the protein content in spirulina can be influenced by cultivation and environmental conditions, e.g., light intensity, climate, and water/air quality, here the qualitative chemical profiles of the examined samples were characterized by similar composition in high-quality proteins as phycocyanins and phycoerythrins.

Atmospheric water-soluble organic carbon (WSOC) is a critical component of airborne particulates. It significantly affects the Earth\'s energy balance, air quality, and human health. Despite its importance, the molecular composition and sources of WSOC remain unclear, particularly in non-urban areas. In this study, we collected total suspended particulate (TSP) samples from three sites in northern China: Erenhot (remote site), Zhangbei (rural site), and Jinan (urban site). The WSOC components were analyzed using high-performance liquid chromatography coupled with high-resolution mass spectrometry. The results showed that the formula numbers of identified compounds exhibited a decreasing trend of Jinan (2647) > Zhangbei (2046) > Erenhot (1399). Among the assigned formulas, CHO compounds were the most abundant category for all three sites, accounting for 33 %-38 % of the identified compounds, followed by the CHON compounds with contributions of 27 %-30 %. In the remote site of Erenhot, CHO compounds were dominated by oxidized unsaturated organic compounds, and CHON compounds were mainly low-oxygenated aliphatic compounds, suggesting a significant influence of primary emissions. In contrast, the urban site of Jinan showed higher contributions of CHO and CHON compounds with elevated oxidation degrees, indicating the influence of more extensive secondary oxidation processes. Atmospheric WSOC in Erenhot and Zhangbei had abundant reduced sulfur-containing species, likely from coal or diesel combustion, while that in Jinan was characterized by aliphatic organosulfates and nitrooxy-organosulfates, which are mainly associated with traffic emissions and biogenetic sources, respectively. These findings reveal significant differences in the molecular composition of WSOC in different atmospheric environments and improve our understanding of the chemical properties, potential sources, and transformations of organic aerosols.

Biosolids are a byproduct of wastewater treatment that can be beneficially applied to agricultural land as a fertilizer. While U.S. regulations limit metals and pathogens in biosolids intended for land applications, no organic contaminants are currently regulated. Novel techniques can aid in detection, evaluation, and prioritization of biosolid-associated organic contaminants (BOCs). For example, nontargeted analysis (NTA) can detect a broad range of chemicals, producing data sets representing thousands of measured analytes that can be combined with computational toxicological tools to support human and ecological hazard assessment and prioritization. We combined NTA with a computer-based tool from the U.S. EPA, the Cheminformatics Hazard Comparison Module (HCM), to identify and prioritize BOCs present in U.S. and Canadian biosolids (n = 16). Four-hundred fifty-one features were detected in at least 80% of samples, with identities of 92 compounds confirmed or assigned probable structures. These compounds were primarily categorized as endogenous compounds, pharmaceuticals, industrial chemicals, and fragrances. Examples of top prioritized compounds were p-cresol and chlorophene, based on human health end points, and fludioxonil and triclocarban, based on ecological health end points. Combining NTA results with hazard comparison data allowed us to prioritize compounds to be included in future studies of the environmental fate and transport of BOCs.

Pollutants in human milk are critical for evaluating maternal internal exposure and infant external exposure. However, most studies have focused on a limited range of pollutants. Here, 15 pooled samples (prepared from 467 individual samples) of human milk from three areas of the Yangtze River Delta (YRD) in China were analyzed by gas chromatography quadrupole time-of-flight mass spectrometry. In total, 171 compounds of nine types were preliminarily identified. Among these, 16 compounds, including 2,5-di-tert-butylhydroquinone and 2-tert-butyl-1,4-benzoquinone, were detected in human milk for the first time. Partial least-squares discriminant analysis identified ten area-specific pollutants, including 2-naphthylamine, 9-fluorenone, 2-isopropylthianthrone, and benzo[a]pyrene, among pooled human milk samples from Shanghai (n = 3), Jiangsu Province (n = 6), and Zhejiang Province (n = 6). Risk index (RI) values were calculated and indicated that legacy polycyclic aromatic hydrocarbons (PAHs) contributed only 20% of the total RIs for the identified PAHs and derivatives, indicating that more attention should be paid to PAHs with various functional groups. Nine priority pollutants in human milk from the YRD were identified. The most important were 4-tert-amylphenol, caffeine, and 2,6-di-tert-butyl-p-benzoquinone, which are associated with apoptosis, oxidative stress, and other health hazards. The results improve our ability to assess the health risks posed by pollutants in human milk.

The popularity of plant-based meat alternatives (PBMAs) has sparked a contentious debate about their influence on intestinal homeostasis compared to traditional animal-based meats. This study aims to explore the changes in gut microbial metabolites (GMMs) induced by the gut microbiota on different digested patties: beef meat and pea-protein PBMA. After digesting in vitro, untargeted metabolomics revealed 32 annotated metabolites, such as carnitine and acylcarnitines correlated with beef meat, and 45 annotated metabolites, like triterpenoids and lignans, linked to our PBMA. Secondly, (un)targeted approaches highlighted differences in GMM patterns during colonic fermentations. Our findings underscore significant differences in amino acids and their derivatives. Beef protein fermentation resulted in higher production of methyl-histidine, gamma-glutamyl amino acids, indoles, isobutyric and isovaleric acids. In contrast, PBMAs exhibit a significant release of N-acyl amino acids and unique dipeptides, like phenylalanine-arginine. This research offers valuable insights into how PBMAs and animal-based proteins differently modulate intestinal microenvironments.

The analysis of pesticide residues and mycotoxins in baby food demands exceptionally low limits of quantitation, necessitating the use of highly sensitive instruments capable of conducting trace analyses. High-resolution instruments typically fail to detect such low levels. However, the latest advancements in liquid time-of-flight technology, when coupled with ion trapping, enable ion enrichment, thereby improving detection levels. This allows for the analysis of these substances at low concentration levels, benefiting from enhanced mass accuracy. Additionally, the use of mass accuracy data helped eliminate matrix interferences, thereby enabling high-confidence identification. We developed a multi-residue method to analyse 219 pesticide residues and 9 mycotoxin residues in baby food matrices. Utilizing a QuEChERS-based extraction method, the samples were then analysed using an LC-Zeno® trap QTOF with mass window screening acquisition. For pesticides, the limit of quantitation was 0.001-0.003 mg/kg for 81 % of the evaluated compounds, 0.005 mg/kg for 13 %, 0.010 mg/kg for 4 % and 0.020-0.030 for 2 %; good linearities were obtained at these levels. Apparent recoveries were evaluated at 0.003, 0.005, and 0.010 mg/kg. At the lowest recovery level, 93 % of compounds showed recoveries between 70 and 120 %. The rest of the compounds were in the range of 63-129 %, with relative standard deviation values below 20 %. For mycotoxins, the limits of quantitation ranged from 0.0001 to 0.100 mg/kg, with matrix-matched concentrations assessed within this range. Recoveries were evaluated at low concentration range (0.001-0.003 mg/kg) and high range (0.020-0.050) with apparent recoveries values between 92 and 140 %. Finally, a total of 31 commercial baby food samples were analysed using this method. The results indicated that 16 samples contained pesticide residues, while two samples were found to have mycotoxins.

Due to the decoupling of the first (1D) and second (2D) dimension in pulsed elution-LC × LC (PE-LC × LC), method development is more flexible and straightforward compared to fast comprehensive LC × LC where the dependencies of key parameters between the two dimensions limits its flexibility. In this study we present a method for pulse generation, which is based on a switching valve alternating between one pump that delivers the gradient and a second pump that delivers low eluotrophic strength for the pause state. Consequently, the dwell volume of the system was circumvented and 7.5, and 3.75 times shorter pulse widths could be generated at flow rates of 0.2, and 0.4 mL/min with satisfactory accuracies between programmed and observed mobile phase composition (relative deviation of 6.0 %). We investigated how key parameters including pulse width and step height, 2D gradient time and flow rate affected the peak capacity in PE-LC × LC. The conditions yielding the highest peak capacity for the PE-LC × LC- high-resolution mass spectrometry (HRMS) system were applied to a wastewater effluent sample. The results were compared to a one dimensional (1D)-LC-HRMS chromatogram. The peak capacity increased with a factor 34 from 112 for the 1D-LC run to 3770 for PE-LC × LC-HRMS after correction for undersampling. The analysis time for PE-LC × LC-HRMS was 12.1 h compared to 67.5 min for the 1D-LC-HRMS run. The purity of the mass spectra improved for PE-LC × LC-HRMS by a factor 2.6 (p-value 3.3 × 10-6) and 2.0 (p-value 2.5 × 10-3) for the low and high collision energy trace compared to the 1D-LC-HRMS analysis. Furthermore, the signal-to-noise ratio (S/N) was 4.2 times higher (range: 0.06-56.7, p-value 3.8 × 10-2) compared to the 1D-LC-HRMS separation based on 42 identified compounds. The improvements in S/N were explained by the lower peak volume obtained in the PE-LC × LC-HRMS.

Lignin is a promising renewable source of valuable organic compounds and environmentally benign materials. However, its involvement in economic circulation and the creation of new biorefining technologies require an understanding of its chemical composition and structure. This problem can be overcome by applying mass spectrometry analytical techniques in combination with advanced chemometric methods for mass spectra processing. The present study is aimed at the development of mass defect filtering to characterize the chemical composition of lignin at the molecular level. This study introduces a novel approach involving resolution-enhanced Kendrick mass defect (REKMD) analysis for the processing of atmospheric pressure photoionization Orbitrap mass spectra of lignin. The set of priority Kendrick fractional base units was predefined in model experiments and provided a substantially expanding available mass defect range for the informative visualization of lignin mass spectra. The developed REKMD analysis strategy allowed to obtain the most complete data on all the homologous series typical of lignin and thus facilitated the interpretation and assignment of elemental compositions and structural formulas to oligomers detected in extremely complex mass spectra, including tandem ones. For the first time, the minor modifications (sulfation) of lignin obtained in ionic liquid-based biorefining processes were revealed.