Metabolic Circuits in Sap Extracts Reflect the Effects of a Microbial Biostimulant on Maize Metabolism under Drought Conditions.

The use of microbial biostimulants in the agricultural sector is increasingly gaining momentum and drawing scientific attention to decode the molecular interactions between the biostimulants and plants. Although these biostimulants have been shown to improve plant health and development, the underlying molecular phenomenology remains enigmatic. Thus, this study is a metabolomics work to unravel metabolic circuits in sap extracts from maize plants treated with a microbial biostimulant, under normal and drought conditions. The biostimulant, which was a consortium of different Bacilli strains, was applied at the planting stage, followed by drought stress application. The maize sap extracts were collected at 5 weeks after emergence, and the extracted metabolites were analyzed on liquid chromatography-mass spectrometry platforms. The acquired data were mined using chemometrics and bioinformatics tools. The results showed that under both well-watered and drought stress conditions, the application of the biostimulant led to differential changes in the profiles of amino acids, hormones, TCA intermediates, phenolics, steviol glycosides and oxylipins. These metabolic changes spanned several biological pathways and involved a high correlation of the biochemical as well as structural metabolic relationships that coordinate the maize metabolism. The hypothetical model, postulated from this study, describes metabolic events induced by the microbial biostimulant for growth promotion and enhanced defences. Such understanding of biostimulant-induced changes in maize sap pinpoints to the biochemistry and molecular mechanisms that govern the biostimulant-plant interactions, which contribute to ongoing efforts to generate actionable knowledge of the molecular and physiological mechanisms that define modes of action of biostimulants.

Combining High-Resolution Gas Chromatographic Continuous Fraction Collection with Nuclear Magnetic Resonance Spectroscopy: Possibilities of Analyzing a Whole GC Chromatogram.

This study presents, for the first time, the successful application of analyzing a whole gas chromatography (GC) chromatogram by nuclear magnetic resonance (NMR) spectroscopy using a continuous repeatable and stable (n = 280) high-resolution (HR) GC fractionation platform with a 96-well plate. Typically with GC- or liquid chromatography-mass spectrometry analysis, (isomer) standards and/or additional NMR analysis are needed to confirm the identification and/or structure of the analyte of interest. In the case of complex substances (e.g., UVCBs), isomer standards are often unavailable and NMR spectra too complex to achieve this. This proof of concept study shows that a HR GC fractionation collection platform was successfully applied to separate, purify, and enrich isomers in complex substances from a whole GC chromatogram, which would facilitate NMR analysis. As a model substance, a chlorinated paraffin (CP) mixture (>8,000 isomers) was chosen. NMR spectra were obtained from all 96 collected fractions, which provides important information for unravelling their full structure. As a proof of concept, a spectral interpretation of a few NMR spectra was made to assign sub-structures. More research is ongoing for the full characterization of CP isomers using multivariate statistical analysis. For the first time, up to only a few CP isomers per fraction were isolated from a highly complex mixture. These may be further purified and certified as standards, which are urgently needed, and can also be used for persistency, bioaccumulation, or toxicity studies.

Identification of mutagenic and endocrine disrupting compounds in surface water and wastewater treatment plant effluents using high-resolution effect-directed analysis.

Effect-directed analysis (EDA) has shown its added value for the detection and identification of compounds with varying toxicological properties in water quality research. However, for routine toxicity assessment of multiple toxicological endpoints, current EDA is considered labor intensive and time consuming. To achieve faster EDA and identification, a high-throughput (HT) EDA platform, coupling a downscaled luminescent Ames and cell-based reporter gene assays with a high-resolution fraction collector and UPLC-QTOF MS, was developed. The applicability of the HT-EDA platform in the analysis of aquatic samples was demonstrated by analysis of extracts from WWTP influent, effluent and surface water. Downscaled assays allowed detection of mutagenicity and androgen, estrogen and glucocorticoid agonism following high-resolution fractionation in 228 fractions. From 8 masses tentatively identified through non-target analysis, 2 masses were further investigated and chemically and biologically confirmed as the mutagen 1,2,3-benzotriazole and the androgen androstenedione. The compatibility of the high-throughput EDA platform with analysis of water samples and the incorporation of mutagenic and endocrine disruption endpoints allow for future application in routine monitoring in drinking water quality control and improved identification of (emerging) mutagens and endocrine disruptors.

Compound Identification Using Liquid Chromatography and High-Resolution Noncontact Fraction Collection with a Solenoid Valve.

We describe the development of a high-resolution, noncontact fraction collector for liquid chromatography (LC) separations, allowing high-resolution fractionation in high-density well plates. The device is based on a low-dead-volume solenoid valve operated at 1-30 Hz for accurate collection of fractions of equal volume. The solenoid valve was implemented in a modified autosampler resulting in the so-called FractioMate fractionator. The influence of the solenoid supply voltage on solvent release was determined and the effect of the frequency, flow rate, and mobile phase composition was studied. For this purpose, droplet release was visually assessed for a wide range of frequencies and flow rates, followed by quantitative evaluation of a selection of promising settings for highly accurate, repeatable, and stable fraction collection. The potential of the new fraction collector for LC-based bioactivity screening was demonstrated by fractionating the LC eluent of a mixture of estrogenic and androgenic compounds, and a surface water sample (blank and spiked with bioactives) combining mass spectrometric detection and two reporter gene assays for bioactivity detection of the fractions. Additionally, a mixture of two compounds was repeatedly LC separated and fractionated to assess the feasibility of the system for analyte isolation followed by nuclear magnetic resonance analysis.

Bisphenol A alternatives in thermal paper from the Netherlands, Spain, Sweden and Norway. Screening and potential toxicity.

Thermal paper contains potentially toxic additives, such as bisphenol A (BPA), as a common color developer. Because of its known endocrine disrupting effects, structural analogues to BPA, such as bisphenol S (BPS), D-8 and Pergafast 201, have been used as alternatives, but little is known about the presence and toxicological effects of alternatives other than BPS. In this study, thermal paper is screened by direct probe ambient mass spectrometry (rapid pre-screening method not requiring sample preparation) and by liquid chromatography (LC) with high resolution time-of flight (TOF-MS) mass spectrometry. Cash receipts and other thermal paper products (cinema tickets, boarding passes and luggage tags) were analyzed. Besides BPA and BPS, other developers only recently reported (Pergafast 201, D-8) or to the best of our knowledge not reported before (D-90, TGSA, BPS-MAE) were frequently found as well as some related unreported impurities (2,4-BPS that is a BPS related impurity and a TGSA related impurity). To gain some insight into the potential estrogenicity of the detected developers, a selection of extracts was further analyzed using a LC-nanofractionation platform in combination with cell-based bioassay testing. These preliminary results seems to indicate very low or absence of estrogenic activity for Pergafast 201, D-8, D-90, TGSA and BPS-MAE in comparison to BPA and BPS, although further dose-response tests with authentic standards are required to confirm these results. Compounds for which standards were available were also tested for developmental toxicity and neurotoxicity using zebrafish (Danio rerio) embryos. TGSA and D-8 induced similar teratogenic effects as BPA in zebrafish embryos. BPS and 2,4-BPS did not induce any developmental effects but 2,4-BPS did alter the locomotor activity at the tested concentration. Our findings suggest that the alternatives used as alternatives to BPA (except BPS) might not be estrogenic. However, TGSA and D-8 showed abnormal developmental effects similar to BPA.

Continuous fraction collection of gas chromatographic separations with parallel mass spectrometric detection applied to cell-based bioactivity analysis.

We describe the development and evaluation of a GC-MS fractionation platform that combines high-resolution fraction collection of full chromatograms with parallel MS detection. A y-split at the column divides the effluent towards the MS detector and towards an inverted y-piece where vaporized trap solvent is infused. The latter flow is directed outside the GC oven allowing subsequent condensation and stepwise collection of liquid fractions with trapped analytes on a 384-well plate. For study and optimization of the effluent split ratio, restriction capillaries of different lengths and diameters were evaluated. For a wide range of settings, local pressures were monitored during fractionation to assess the influence of MS vacuum and trap solvent infusion on the GC system stability. The platform performance was evaluated by GC-MS analysis and continuous fractionation of an n-alkane mixture followed by GC analysis of each fraction. Comparison of the on-line recorded and fraction-reconstructed chromatogram showed the GC separation is maintained during fractionation. Multiple fractionation cycles of the n-alkane sample on the same 384-well plate yielded a reconstructed chromatogram which was highly similar to that of a single analysis, demonstrating the high repeatability. The applicability of the GC-MS-fractionation platform for bioactivity screening was investigated by applying the AR-Ecoscreen reporter gene bioassay on fractions obtained after analysis of standard solutions and dust samples containing the anti-androgenic compounds vinclozolin and p,p'-DDE.

Identification of Novel Brominated Compounds in Flame Retarded Plastics Containing TBBPA by Combining Isotope Pattern and Mass Defect Cluster Analysis.

The study of not only main flame retardants but also of related degradation products or impurities has gained attention in the last years and is relevant to assess the safety of our consumer products and the emission of potential contaminants into the environment. In this study, we show that plastics casings of electric/electronic devices containing TBBPA contain also a complex mixture of related brominated chemicals. These compounds were most probably coming from impurities, byproducts, or degradation products of TBBPA and TBBPA derivatives. A total of 14 brominated compounds were identified based on accurate mass measurements (formulas and tentative structures proposed). The formulas (or number of bromine elements) for 19 other brominated compounds of minor intensity are also provided. A new script for the recognition of halogenated compounds based on combining a simplified isotope pattern and mass defect cluster analysis was developed in R for the screening. The identified compounds could be relevant from an environmental and industrial point of view.

Highly Selective Screening of Estrogenic Compounds in Consumer-Electronics Plastics by Liquid Chromatography in Parallel Combined with Nanofractionation-Bioactivity Detection and Mass Spectrometry.

The chemical safety of consumer products is an issue of emerging concern. Plastics are widely used, e.g. as casings of consumer electronics (TVs, computers, routers, etc.), which are present in houses and offices in continuously increasing numbers. In this study, we investigate the estrogenic activity of components of plastics coming from electronics' casings. A recently developed fractionation platform for effect-directed analysis (EDA) was used. This platform combines reversed-phase liquid chromatography in parallel with bioassay detection via nanofractionation and with online high-resolution time-of-flight mass spectrometry (TOFMS) for the identification of bioactives. Four out of eight of the analyzed plastics samples showed the presence of estrogenic compounds. Based on the MS results these were assigned to bisphenol A (BPA), 2,4-di-tert-butylphenol, and a possible bisphenol A analog. All samples contained flame retardants, but these did not show any estrogenicity. The observed BPA, however, could be an impurity of tetrabromo-BPA (TBBPA) or TBBPA-based flame retardants. Due to the plausible migration of additives from plastics into the environment, plastics from consumer electronics likely constitute a source of estrogenic compound contamination in the indoor environment.

Gas chromatography fractionation platform featuring parallel flame-ionization detection and continuous high-resolution analyte collection in 384-well plates.

Gas chromatography (GC) is a superior separation technique for many compounds. However, fractionation of a GC eluate for analyte isolation and/or post-column off-line analysis is not straightforward, and existing platforms are limited in the number of fractions that can be collected. Moreover, aerosol formation may cause serious analyte losses. Previously, our group has developed a platform that resolved these limitations of GC fractionation by post-column infusion of a trap solvent prior to continuous small-volume fraction collection in a 96-wells plate (Pieke et al., 2013 [17]). Still, this GC fractionation set-up lacked a chemical detector for the on-line recording of chromatograms, and the introduction of trap solvent resulted in extensive peak broadening for late-eluting compounds. This paper reports advancements to the fractionation platform allowing flame ionization detection (FID) parallel to high-resolution collection of a full GC chromatograms in up to 384 nanofractions of 7s each. To this end, a post-column split was incorporated which directs part of the eluate towards FID. Furthermore, a solvent heating device was developed for stable delivery of preheated/vaporized trap solvent, which significantly reduced band broadening by post-column infusion. In order to achieve optimal analyte trapping, several solvents were tested at different flow rates. The repeatability of the optimized GC fraction collection process was assessed demonstrating the possibility of up-concentration of isolated analytes by repetitive analyses of the same sample. The feasibility of the improved GC fractionation platform for bioactivity screening of toxic compounds was studied by the analysis of a mixture of test pesticides, which after fractionation were subjected to a post-column acetylcholinesterase (AChE) assay. Fractions showing AChE inhibition could be unambiguously correlated with peaks from the parallel-recorded FID chromatogram.

Effect-directed analysis supporting monitoring of aquatic environments--An in-depth overview.

Aquatic environments are often contaminated with complex mixtures of chemicals that may pose a risk to ecosystems and human health. This contamination cannot be addressed with target analysis alone but tools are required to reduce this complexity and identify those chemicals that might cause adverse effects. Effect-directed analysis (EDA) is designed to meet this challenge and faces increasing interest in water and sediment quality monitoring. Thus, the present paper summarizes current experience with the EDA approach and the tools required, and provides practical advice on their application. The paper highlights the need for proper problem formulation and gives general advice for study design. As the EDA approach is directed by toxicity, basic principles for the selection of bioassays are given as well as a comprehensive compilation of appropriate assays, including their strengths and weaknesses. A specific focus is given to strategies for sampling, extraction and bioassay dosing since they strongly impact prioritization of toxicants in EDA. Reduction of sample complexity mainly relies on fractionation procedures, which are discussed in this paper, including quality assurance and quality control. Automated combinations of fractionation, biotesting and chemical analysis using so-called hyphenated tools can enhance the throughput and might reduce the risk of artifacts in laboratory work. The key to determining the chemical structures causing effects is analytical toxicant identification. The latest approaches, tools, software and databases for target-, suspect and non-target screening as well as unknown identification are discussed together with analytical and toxicological confirmation approaches. A better understanding of optimal use and combination of EDA tools will help to design efficient and successful toxicant identification studies in the context of quality monitoring in multiply stressed environments.

Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry.

In this study we developed a new LC nanofractionation platform that combines a human cell (BG1.Luc) gene reporter assay with a high resolution mass spectrometer for the detection and identification of estrogenic and anti-estrogenic compounds in environmental waters. The selection of this assay was based on its high sensitivity and selectivity, which is required for environmental trace level detection. We modified an autosampler and controlled it with in-house developed software to collect fractions in the low second range in microtiter plates. This ensured that chromatographic separation was maintained and allowed straightforward hyphenation with the bioassay. After bioassay testing, bioassay chromatograms were reconstructed and directly correlated with MS chromatograms that were obtained in parallel. This enabled to pinpoint bioactives in the MS chromatogram within a single fractionation cycle and results in a significant increase in throughput compared to traditional EDA studies. The sensitivity of the platform was low enough for environmental waters (80nM for bisphenol A and 320pM and 3.2nM for estradiol and estriol, respectively). In addition, the ability of the platform to detect anti-estrogens was successfully demonstrated as well. Finally, real samples were analysed.

A hypomorphic Cbx3 allele causes prenatal growth restriction and perinatal energy homeostasis defects.

Mammals have three HP1 protein isotypes HP1 beta (CBX1), HP1 alpha (CBX3) and HP1 alpha (CBX5) that are encoded by the corresponding genes Cbx1, Cbx3 and Cbx5. Recent work has shown that reduction of CBX3 protein in homozygotes for a hypomorphic allele (Cbx3hypo) causes a severe postnatal mortality with around 99 percent of the homozygotes dying before weaning. It is not known what the causes of the postnatal mortality are. Here we show that Cbx3hypo/hypo conceptuses are significantly reduced in size and the placentas exhibit a haplo-insufficiency. Late gestation Cbx3hypo/hypo placentas have reduced mRNA transcripts for genes involved in growth regulation, amino acid and glucose transport. Blood vessels within the Cbx3hypo/hypo placental labyrinth are narrower than wild-type. Newborn Cbx3hypo/hypo pups are hypoglycemic, the livers are depleted of glycogen reserves and there is almost complete loss of stored lipid in brown adipose tissue (BAT). There is a 10-fold reduction in expression of the BAT-specific Ucp1 gene, whose product is responsible for nonshivering themogenesis. We suggest that it is the small size of the Cbx3hypo/hypo neonates, a likely consequence of placental growth and transport defects, combined with a possible inability to thermoregulate that causes the severe postnatal mortality.

Detection and structural elucidation of esterified oxylipids in human synovial fluid by electrospray ionization-fourier transform ion-cyclotron mass spectrometry and liquid chromatography-ion trap-MS(3): detection of esterified hydroxylated docosapentaenoic acid containing phospholipids.

Here, we present the application of a cross-platform approach, combining rapid direct infusion high-resolution/accurate mass electrospray ionization Fourier transform ion-cyclotron mass spectrometry (ESI-FTICRMS) with in-depth data-dependent LC-MS(2) and LC-MS(3) analysis for lipid profiling. The analytical approach as well as the subsequent data handling is described. The method was applied to human synovial fluid samples from osteo- and rheumatoid arthritis patients. Multivariate statistical analysis revealed esterified oxylipids as molecular features in a subset of the patient samples. Employing LC-MS(2) and LC-MS(3) analysis of these species, we were able to clarify the hypothesized lipid structures initially based on the accurate mass measurements performed on the ESI-FTICRMS platform. LC-MS(3) analysis of intact esterified oxy-lipids and LC-MS(2) analysis of the hydrolysis products allowed for the detection of positional isomers. The approach led to the structural elucidation of hydroxylated docosapentaenoic acid-containing diacyl-phosphatidylcholine type phospholipids in human synovial fluid.

Blood plasma sample preparation method for the assessment of thyroid hormone-disrupting potency in effect-directed analysis.

A sample preparation method combining solid-phase extraction (SPE) and liquid-liquid extraction (LLE) was developed to be used in Effect-Directed Analysis (EDA) of blood plasma. Until now such a method was not available. It can be used for extraction of a broad range of thyroid hormone (TH)-disruptors from plasma with high recoveries. Validation of the method using spiked cow plasma showed good recoveries for hydroxylated polybrominated diphenyl ethers (OH-PBDEs; 93.8 ± 19.5%), hydroxylated polychlorinated biphenyls (OH-PCBs; 93.8 ± 15.5%), other halogenated phenols (OHPs; 107 ± 8.1%), and for short-chain (<8 C-atoms) perfluoroalkyl substances (PFASs; 85.2 ± 24.6%). In the same extracts, the potency of the compound classes spiked to the cow plasma to competitively bind to transthyretin (TTR) was recovered by 84.9 ± 8.8%. Furthermore, the SPE-LLE method efficiently removed endogenous THs from the extracts, thereby eliminating their possible contribution to the binding assay response. The SPE-LLE method was applied to polar bear plasma samples to investigate its applicability in future EDA studies focusing on TH-disrupting compounds in this top predator species that is exposed to relatively high levels of bioaccumulating pollutants. A first screening revealed TTR-binding potency in the polar bear plasma extracts, which could be explained for 60-85% by the presence of OH-PCBs.