Few human data on exposure and toxicity are available on neonicotinoids and neonicotinoid-like compounds (NNIs), an important group of insecticides worldwide. Specifically, exposure assessment of ...humans by biomonitoring remains a challenge due to the lack of appropriate biomarkers. We investigated the human metabolism and metabolite excretion in urine of acetamiprid (ACE), clothianidin (CLO), flupyradifurone (FLUP), imidacloprid (IMI), sulfoxaflor (SULF), thiacloprid (THIAC) and thiamethoxam (THIAM) after single oral dosages at the currently acceptable daily intake levels of the European Food Safety Authority. Consecutive post-dose urine samples were collected up to 48 h. Suspect screening of tentative metabolites was carried out by liquid chromatography–high-resolution mass spectrometry. Screening hits were identified based on their accurate mass, isotope signal masses and ratios, product ion spectra, and excretion kinetics. We found, with the exception of SULF, extensive metabolization of NNIs to specific metabolites which were excreted next to the parent compounds. Overall, 24 metabolites were detected with signal intensities indicative of high metabolic relevance. Phase-I metabolites were predominantly derived by mono-oxidation (such as hydroxy-FLUP, -IMI, and -THIAC) and by oxidative
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-desalkylation (such as
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-desdifluoroethyl-FLUP and
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-desmethyl-ACE, -CLO and -THIAM). IMI-olefin, obtained by dehydration of hydroxylated IMI, was identified as a major metabolite of IMI. SULF was excreted unchanged in urine. Previously reported metabolites of NNIs such as 6-chloronicotinic acid or 2-chlorothiazole-4-carboxylic acid and their glycine derivatives were detected either at low signal intensities or not at all and seem less relevant for human biomonitoring. Our highly controlled approach provides specific insight into the human metabolism of NNIs and suggests suitable biomarkers for future exposure assessment at environmentally relevant exposures.
Molecular networking connects mass spectra of molecules based on the similarity of their fragmentation patterns. However, during ionization, molecules commonly form multiple ion species with ...different fragmentation behavior. As a result, the fragmentation spectra of these ion species often remain unconnected in tandem mass spectrometry-based molecular networks, leading to redundant and disconnected sub-networks of the same compound classes. To overcome this bottleneck, we develop Ion Identity Molecular Networking (IIMN) that integrates chromatographic peak shape correlation analysis into molecular networks to connect and collapse different ion species of the same molecule. The new feature relationships improve network connectivity for structurally related molecules, can be used to reveal unknown ion-ligand complexes, enhance annotation within molecular networks, and facilitate the expansion of spectral reference libraries. IIMN is integrated into various open source feature finding tools and the GNPS environment. Moreover, IIMN-based spectral libraries with a broad coverage of ion species are publicly available.
•Cereal lipids were analysed using GC/Q-TOF MS.•Differences in sterol, tocopherol and alkylresorcinol patterns could be observed.•Novel tocomonoenols and methyl-alkylresorcinols were detected.•The ...cereals could be discriminated by PCA based on non-targeted lipid profiles.•Non-targeted lipid profiling could be a new tool for cereal authenticity.
Minor lipids in cereals (such as phytosterols and alkylresorcinols) can be important for human nutrition and/or be used as biomarkers for cereal intake. However, the analysis of cereal lipids is very challenging due to the complex lipidome comprising several hundred individual compounds present over a wide range of concentrations.
Here we present a method for the profiling of cereal lipids using high temperature gas chromatography coupled to high resolution mass spectrometry (GC/Q-TOF MS). The method was used to investigate the lipid profiles of 77 samples of bread wheat, spelt, einkorn, emmer, barley, rye and oats.
Distinct differences in the patterns of alkylresorcinols, free and conjugated sterols and tocopherols between the cereals could be observed. Furthermore, traces of tocomonoenols and diunsaturated and methyl-alkylresorcinols (not previously reported in cereals) could be detected. Finally, the lipid patterns in the cereals could be used to separate the cereals by Principal Component Analysis.
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•Human toxicokinetics of homosalate (HMS, cis and trans) after oral dose.•≥15 oxidative metabolites investigated quantitatively/semi-quantitatively.•≥70 % (average; n = 4) of urinary ...excretion of each metabolite within 24 h post dose.•Urinary excretion fraction sums were 100-fold higher for trans-derived metabolites.•Plasma data indicate 10-fold lower oral bioavailability of cis-homosalate.
Homosalate (HMS) is a salicylate UV filter broadly used in sunscreens and personal care products. The aim of this study was the collection of human toxicokinetic data on HMS as a tool for risk assessment. For this purpose, metabolism and urinary excretion after a single oral HMS dose (98.2–149.1 µg (kg body weight)−1) were investigated in four volunteers (two male, two female). As commercial products generally contain a mixture of cis- and trans-HMS, both cis-rich and trans-rich isomer mixtures were studied to investigate possible differences in metabolism. Initial metabolite screening tentatively identified six oxidative metabolite subgroups, of which hydroxylated and carboxylic acid metabolites were studied in more detail. Unchanged parent HMS and the previously identified HMS metabolites 5-((2-hydroxybenzoyl)oxy)-3,3-dimethylcyclohexane-1-carboxylic acid (HMS-CA) and 3-hydroxy-3,5,5-trimethylcyclohexyl 2-hydroxybenzoate (3OH-HMS), respectively, were quantified separately as cis- and trans-isomers via authentic standards by isotope dilution analysis. In addition, further alkyl-hydroxylated and carboxylic acid metabolites were investigated semi-quantitatively. Peak concentrations in urine were reached 1.5–6.3 h post-dose and more than 80 % of each of the quantitatively investigated metabolites (and at least 70 % of the semi-quantitatively investigated metabolites) was excreted within the first 24 h. Plasma and urine data indicated that oral bioavailability of cis-HMS was one order of magnitude below that of trans-HMS. Furthermore, the mean total urinary excretion fraction (Fue) for the metabolites derived from trans-HMS (6.4 %) was two orders of magnitude higher than for the metabolites derived from cis-HMS (0.045 %). Our data proves diastereoselectivity in toxicokinetics of cis- and trans-HMS, emphasizing the necessity to address isomer ratios in future studies including HMS exposure and risk assessments.
Lipids are biomolecules with a broad variety of chemical structures, which renders them essential not only for various biological functions but also interestingly for biotechnological applications. ...Rhamnolipids are microbial glycolipids with surface-active properties and are widely used biosurfactants. They are composed of one or two
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-rhamnoses and up to three hydroxy fatty acids. Their biosynthetic precursors are 3-hydroxy(alkanoyloxy)alkanoic acids (HAAs). The latter are also present in cell supernatants as complex mixtures and are extensively studied for their potential to replace synthetically derived surfactants. The carbon chain lengths of HAAs determine their physical properties, such as their abilities to foam and emulsify, and their critical micelle concentration. Despite growing biotechnological interest, methods for structural elucidation are limited and often rely on hydrolysis and analysis of free hydroxy fatty acids losing the connectivity information. Therefore, a high-performance liquid chromatography-mass spectrometry method was developed for comprehensive structural characterization of intact HAAs. Information is provided on chain length and number of double bonds in each hydroxy fatty acid and their linkage by tandem mass spectrometry (MS/MS). Post-column photochemical derivatization by online Paternὸ–Büchi reaction and MS/MS fragmentation experiments generated diagnostic fragments allowing structural characterization down to the double bond position level. Furthermore, the presented experiments demonstrate a powerful approach for structure elucidation of complex lipids by tailored fragmentation.
Rhamnolipids are biosurfactants featuring surface-active properties that render them suitable for a broad range of industrial applications. These properties include their emulsification and foaming ...capacity, critical micelle concentration, and ability to lower surface tension. Further, aspects like biocompatibility and environmental friendliness are becoming increasingly important. Rhamnolipids are mainly produced by pathogenic bacteria like Pseudomonas aeruginosa. We previously designed and constructed a recombinant Pseudomonas putida KT2440, which synthesizes rhamnolipids by decoupling production from host-intrinsic regulations and cell growth.
Here, the molecular structure of the rhamnolipids, i.e., different congeners produced by engineered P. putida are reported. Natural rhamnolipid producers can synthesize mono- and di-rhamnolipids, containing one or two rhamnose molecules, respectively. Of each type of rhamnolipid four main congeners are produced, deviating in the chain lengths of the β-hydroxy-fatty acids. The resulting eight main rhamnolipid congeners with variable numbers of hydrophobic/hydrophilic residues and their mixtures feature different physico-chemical properties that might lead to diverse applications. We engineered a microbial cell factory to specifically produce three different biosurfactant mixtures: a mixture of di- and mono-rhamnolipids, mono-rhamnolipids only, and hydroxyalkanoyloxy alkanoates, the precursors of rhamnolipid synthesis, consisting only of β-hydroxy-fatty acids. To support the possibility of second generation biosurfactant production with our engineered microbial cell factory, we demonstrate rhamnolipid production from sustainable carbon sources, including glycerol and xylose. A simple purification procedure resulted in biosurfactants with purities of up to 90%. Finally, through determination of properties specific for surface active compounds, we were able to show that the different mixtures indeed feature different physico-chemical characteristics.
The approach demonstrated here is a first step towards the production of designer biosurfactants, tailor-made for specific applications by purposely adjusting the congener composition of the mixtures. Not only were we able to genetically engineer our cell factory to produce specific biosurfactant mixtures, but we also showed that the products are suited for different applications. These designer biosurfactants can be produced as part of a biorefinery from second generation carbon sources such as xylose.
An instrumental method for the evaluation of olive oil quality was developed. Twenty-one relevant aroma active compounds were quantified in 95 olive oil samples of different quality by headspace ...solid phase microextraction (HS-SPME) and dynamic headspace coupled to GC-MS. On the basis of these stable isotope dilution assay results, statistical evaluation by partial least-squares discriminant analysis (PLS-DA) was performed. Important variables were the odor activity values of ethyl isobutanoate, ethyl 2-methylbutanoate, 3-methylbutanol, butyric acid, E,E-2,4-decadienal, hexanoic acid, guaiacol, 2-phenylethanol, and the sum of the odor activity values of Z-3-hexenal, E-2-hexenal, Z-3-hexenyl acetate, and Z-3-hexenol. Classification performed with these variables predicted 88% of the olive oils’ quality correctly. Additionally, the aroma compounds, which are characteristic for some off-flavors, were dissolved in refined plant oil. Sensory evaluation of these models demonstrated that the off-flavors rancid, fusty, and vinegary could be successfully simulated by a limited number of odorants.
•Oxalic acid analysis by means of ion exclusion chromatography-tandem MS.•Quantification with stable isotope dilution approach.•Application in complex matrices like mouse urine or primary mouse ...hepatocyte cell culture supernatants.
Due to medical relevance and a direct correlation with some diseases, accurate quantification of oxalic acid in different complex matrices is required. Effective chromatographic separation of this strong carboxylic acid was achieved by ion exclusion chromatography (IELC). Sensitive and selective detection was carried out by means of electrospray ionization-tandem mass spectrometry. Furthermore, it was shown that the isobaric interference of lactic acid is chromatographically resolved. Structurally similar compounds like glyoxylic acid and glycolic acid were baseline separated as well. The application of stable isotope dilution analysis with 13C2 oxalic acid facilitated precise quantification. The developed method was validated with a reference oxalate sample of human urine diluted to a range of 10–500μM. Finally, the applicability of this method was demonstrated on complex matrices, like mouse urine and supernatants of primary mouse hepatocyte cell cultures.
Rationale
The rising field of lipidomics strongly relies on the identification of lipids in complex matrices. Recent technical advances regarding liquid chromatography (LC) and high‐resolution mass ...spectrometry (HRMS) enable the mapping of the lipidome of an organism with short data acquisition times. However, interpretation and evaluation of resulting multidimensional datasets are challenging and this is still the bottleneck regarding overall analysis times.
Methods
A novel adaption of Kendrick mass plot analysis is presented for a rapid and accurate analysis of lipids in complex matrices. Separation of lipids by their respective head group was achieved via hydrophilic interaction liquid chromatography (HILIC) coupled to HRMS. The resulting LC/HRMS datasets are processed to a list of chromatographically separated features by applying an optimized MZmine 2 workflow. All features are plotted in a three‐dimensional Kendrick mass plot, which allows a fast identification of present lipid classes, based on equidistant features with fitting retention times and the same Kendrick mass defect. Suspected lipid classes are used for exact mass database matching to annotate features. A second three‐dimensional Kendrick mass plot of annotated features of a single lipid class helps to reveal potential database mismatches, resulting in a curated list of identified lipid species.
Results
The use of the novel adaption of the Kendrick mass plot has accelerated the identification of the relevant lipid species in the green alga Chlamydomonas reinhardtii. A total of 106 species were identified within the lipid classes: phosphatidylserine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and sulfoquinovosyldiacylglycerol.
Conclusions
This work shows how the addition of chromatographic information, i.e. the retention time, to a classical two‐dimensional Kendrick mass plot enables rapid and accurate analysis of LC/HRMS datasets, exemplified on a green alga (C. reinhardtii) sample. Three‐dimensional Kendrick mass plots have improved lipid class identification and fast spotting of falsely annotated lipid species.
To broaden the substrate scope of microbial cell factories towards renewable substrates, rational genetic interventions are often combined with adaptive laboratory evolution (ALE). However, ...comprehensive studies enabling a holistic understanding of adaptation processes primed by rational metabolic engineering remain scarce. The industrial workhorse Pseudomonas putida was engineered to utilize the non-native sugar D-xylose, but its assimilation into the bacterial biochemical network via the exogenous xylose isomerase pathway remained unresolved. Here, we elucidate the xylose metabolism and establish a foundation for further engineering followed by ALE. First, native glycolysis is derepressed by deleting the local transcriptional regulator gene hexR. We then enhance the pentose phosphate pathway by implanting exogenous transketolase and transaldolase into two lag-shortened strains and allow ALE to finetune the rewired metabolism. Subsequent multilevel analysis and reverse engineering provide detailed insights into the parallel paths of bacterial adaptation to the non-native carbon source, highlighting the enhanced expression of transaldolase and xylose isomerase along with derepressed glycolysis as key events during the process.
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