Is Fucus a suitable biomonitoring organism for polycyclic aromatic hydrocarbon contamination? A study from the Faroe Islands.

To evaluate seaweed as a biomonitoring organism, Fucus was sampled in the Faroe Islands. Nineteen PAHs, including the EPA 16, and four groups of alkylated PAHs were quantified using GC-MS analysis of extracts obtained using a modified QuEchERS method with ultrasonication in acetonitrile, back-extraction into hexane, and Florisil® cleanup. Samples from the harbor of Tórshavn collected at high tide were the most polluted with PAH concentrations between 1.3 × 102 and 1.7 × 102 ng/g wet weight. All samples contained a factor 10 higher concentrations of alkylated PAHs compared to their parent compounds. These results suggest that Fucus might be suitable as a biomonitoring organism for PAH pollution. Differences between samples collected in close proximity and on different days were observed (same range of RSD 14-120% and 60-102%, respectively), suggesting that water exchange, tide levels, and direct exposure to surface diesel pollution have a strong influence on pollutant uptake in Fucus. The findings stress the need for further evaluation of the sampling strategy.

Investigation of micropollutants in household waste fractions processed by anaerobic digestion: target analysis, suspect- and non-target screening.

Household waste represents a major source of energy, nutrients, and recyclable material. In order to exploit benefits and avoid hazards in the context of circular economy, the risk profile towards human and the environment should be assessed. Here, we investigated the presence of micropollutants by quantitative target analysis, suspect and non-target screening and evaluated changes in the chemical fingerprint upon anaerobic digestion. Extracts were analyzed by reversed phase liquid chromatography high-resolution mass spectrometry (LC-HRMS) and gas chromatography mass spectrometry (GC-MS). Thirty-one of 51 target micropollutants were detected in low ng/mL levels except for few detections at µg/mL levels. The micropollutants quantified in this study included the following: pharmaceuticals (salicylic acid, amitriptyline, carbamazepine); biocides (triclocarban, 2-phenylphenol); industrial compounds used in, e.g., paper industry (pentachlorphenol, PFOS, PFOA, bisphenol A); aromatics, polycyclic aromatics, and heteroaromatics, and their alkylated, hydroxylated, or carboxylated analogues. Fifty of 206 compounds from the suspect screening list were tentatively identified. These included phthalates, methylparaben, phenol, benzophenone, and pharmaceuticals, e.g., ibuprofen. Most compounds detected by GC-MS decreased more than twofold in peak height or remained unaffected by the anaerobic digestion, and very few increased more than twofold, e.g., p-cresol, menthol, and octadecanal. From the LC-HRMS non-target screening analysis, 250 chemical components were resolved using the multiway curve resolution technique PARAFAC2; of these, carbidopa was the only identified unknown.

Generic multicriteria approach to determine the best precipitation agent for removal of biomacromolecules prior to non-targeted metabolic analysis.

The removal of biomacromolecules from biofluids decreases the sample complexity and lower electrospray suppression effects. Furthermore, it can increase the analysis sensitivity, precision, and selectivity. Often removal approaches evaluate the model based on a single criterion, like protein removed or response of one of few specific metabolites. In this study, we used a multicriteria approach to test the effect of using the solvents methanol and acetonitrile (organic solvent precipitation), trichloroacetic acid (acidic precipitation) and ammonium sulphate (salting out) to remove biomacromolecules from a downstream recovery process from a bacillus fermentation. The downstream recovery process intermediates were analysed using reversed-phase ultra-high-pressure liquid chromatography with electrospray ionisation and high-resolution time-of-flight mass spectrometry detection. To evaluate the pre-treatment agents the following multicriteria was applied i) practical considerations, ii) total amino acid in the precipitated pellet, iii) putative identification of the molecules removed or created by the different treatments, iv) coherence between high quality extracted ion chromatograms (repeatability of DW-CODA) and v) replicate consistency from principal component analysis score values obtained by using the CHEMometric analysis of sections of Selected Ion Chromatograms (CHEMSIC) method. This study presents a generic workflow to find the best pre-treatment for removing bio-macromolecules from biofluids with a multicriteria approach. In our case, the best protein removal strategy for downstream recovery intermediates was acetonitrile precipitation. This method showed high precision, created few artefact peaks compared to simple sample dilution, and mainly removed small peptides.

A tiered analytical approach for target, non-target and suspect screening analysis of polar transformation products of polycyclic aromatic compounds.

Polycyclic aromatic compounds (PACs) possess toxicity towards humans, and their presence in the environment is unwanted. Polar transformation products (TPs) are more mobile, and can be considered emerging contaminants, as they represent a more bioavailable carrier of the same toxic properties. Acidic TPs has been proposed as an important class of polar TPs. This study presents a tiered analytical approach to investigate acidic and polar PAC TPs in environmental conditions. The tiered approach exploits target analysis for quantification of acids; suspect screening for tentative identification based on retention time and spectral matching using databases; and finally non-target analysis based on chromatography and data independent broadband MS to highlight potentially unknown analyte peaks. The approach includes a mixed-mode anion exchange solid phase extraction (MAX-SPE) to fractionate neutral and acidic compounds, and is applied to three cases: I) Photo-oxidation of six PACs generated suspected hydroxylated-, carbonylated- and carboxylated PACs but also proposed the presence of mono- and dicarboxylic acids, which have not been reported elsewhere. For a subset of four acids, conversion rates were determined. II) Recovery of spiked acids from diesel spilled harbor water was 80% by LC-MS, and diesel spill weathering was evaluated from the neutral fraction by GC-MS. III) By non-target analysis sulfonated PACs, presumable derived from photo-oxidation, were detected in run-off basins of an arctic landfarm, alongside hypothesized naturally occuring fatty acids. The tiered approach is a sensitive and versatile tool to extract information on PACs and their polar TPs from polluted environmental sites.

Optimizing gradient conditions in online comprehensive two-dimensional reversed-phase liquid chromatography by use of the linear solvent strength model.

The linear solvent strength model was used to predict coverage in online comprehensive two-dimensional reversed-phase liquid chromatography. The prediction model uses a parallelogram to describe the separation space covered with peaks in a system with limited orthogonality. The corners of the parallelogram are assumed to behave like chromatographic peaks and the position of these pseudo-compounds was predicted. A mix of 25 polycyclic aromatic compounds were used as a test. The precision of the prediction, span 0-25, was tested by varying input parameters, and was found to be acceptable with root mean square errors of 3. The accuracy of the prediction was assessed by comparing with the experimental coverages. Less than half of experimental coverages were outside prediction ± 1 × root mean square error and none outside prediction ± 2 × root mean square error. Accuracy was lower when retention factors were low, or when gradient conditions affected parameters not included in the model, e.g. second dimension gradient time affects the second dimension equilibration time. The concept shows promise as a tool for gradient optimization in online comprehensive two-dimensional liquid chromatography, as it mitigates the tedious registration and modeling of all sample constituents, a circumstance that is particularly appealing when dealing with complex samples.

Increasing Flexibility in Two-Dimensional Liquid Chromatography by Pulsed Elution of the First Dimension: A Proof of Concept.

This work demonstrates the development of an online two-dimensional liquid chromatography (2D-LC) method where the first dimension column is eluted by a sequence of pulses of increasing eluotropic strength generated by the LC pumps (pulsed-elution 2D-LC). Between the pulses, the first dimension is kept in a no-elution state using low eluent strength. The eluate from the first dimension is actively modulated using trap columns and subsequently analyzed in the second dimension. We demonstrate that by tuning the length and eluotropic strength of the pulses, peaks with retention factors in water, kw, above 150 can be manipulated to elute in 3-4 pulses. The no-elution state can be kept for 1-10 min with only minor changes as to which and how many pulses the peaks elute in. Pulsed-elution 2D-LC combined with active modulation tackles three of the main challenges encountered in 2D-LC and specifically online comprehensive 2D-LC: undersampling, difficulties in refocusing, and lack of flexibility in the selection of column dimensions and flow rates because the two dimensions constrain each other. The pulsed-elution 2D-LC was applied for the analysis of a basic fraction of vacuum gas oil. Peak capacity was 4018 for a 540 min analysis and 4610 for a 1040 min analysis.

Evaluation of chromatographic conditions in reversed phase liquid chromatography-mass spectrometry systems for fingerprinting of polar and amphiphilic plant metabolites.

Metabolic fingerprinting is a relatively young scientific discipline requiring robust, yet flexible and fit-for-purpose analytical methods. Here, we introduce a simple approach to select reversed phase LC systems with electrospray MS detection for fingerprinting of polar and amphiphilic plant metabolites. The approach does not rely on isotopic labeling or biological origin of sample constituent and can also be used for non-biological matrices (e.g., oil or sewage sludge) or for other optimization purposes (e.g., mass spectrometric source parameterization). The LC systems varied in column chemistry and temperature, mobile phase pH/additive, gradient steepness/eluotropic strength, and electrospray mode of operation. The systems were evaluated based on the number of features detected using the matchedFilter algorithm from XCMS and the repeatability of this detection across analytical replicates. For negative ion mode detection, the best performances were obtained with an HSS T3 column operated at low pH, which produced a 3-fold increase in the number of reliable features extracted compared with the worst system. The best system for positive ion mode (i.e., the BEH C18 column operated at intermediate pH) only produced a 50 % increase in the number of reliable features. The data also indicate that baseline removal is unavoidable for reliable intensity estimations using peak areas, and that peak heights may be a more robust measure of intensity when baselines cannot be completely removed or in case of coelution, fronting or tailing.

Experimental insights into the importance of aquatic bacterial community composition to the degradation of dissolved organic matter.

Bacteria play a central role in the cycling of carbon, yet our understanding of the relationship between the taxonomic composition and the degradation of dissolved organic matter (DOM) is still poor. In this experimental study, we were able to demonstrate a direct link between community composition and ecosystem functioning in that differently structured aquatic bacterial communities differed in their degradation of terrestrially derived DOM. Although the same amount of carbon was processed, both the temporal pattern of degradation and the compounds degraded differed among communities. We, moreover, uncovered that low-molecular-weight carbon was available to all communities for utilisation, whereas the ability to degrade carbon of greater molecular weight was a trait less widely distributed. Finally, whereas the degradation of either low- or high-molecular-weight carbon was not restricted to a single phylogenetic clade, our results illustrate that bacterial taxa of similar phylogenetic classification differed substantially in their association with the degradation of DOM compounds. Applying techniques that capture the diversity and complexity of both bacterial communities and DOM, our study provides new insight into how the structure of bacterial communities may affect processes of biogeochemical significance.

Metabolic fingerprinting of Lactobacillus paracasei: a multi-criteria evaluation of methods for extraction of intracellular metabolites.

An untargeted multi-criteria approach was used to select the best extraction method among freeze-thawing in methanol (FTM), boiling ethanol (BE) and chloroform-methanol (CM) for gas chromatography mass spectrometry (GC-MS) metabolic fingerprinting of Lactobacillus paracasei subsp. paracasei (CRL-431®). The following results were obtained: (i) coverage and efficiency, measured by the number of features extracted and the sum of feature intensities, showed that FTM extraction resulted in the largest compound coverage with a total number of features 8.9 × 10(3) ± 0.5 × 10(3), while merely 6.6 × 10(3) ± 0.9 × 10(3) and 7.9 × 10(3) ± 0.8 × 10(3) were detected in BE or CM, respectively; (ii) the similarity of extraction methods, measured by common features, demonstrated that FTM yielded the most complementary information to BE and CM; i.e. 17 and 33 % of the features of FTM extracted were unique compared to CM and BE, respectively; and (iii) a clear-cut separation according to extraction method was demonstrated by assessment of the metabolic fingerprints by pixel-based data analysis. Indications of metabolite degradation were observed under the elevated temperature for BE extraction. A superior coverage of FTM together with a high repeatability over nearly the whole range of GC-amenable compounds makes this the extraction method of choice for metabolic fingerprinting of L. paracasei.

Using the hydrophobic subtraction model to choose orthogonal columns for online comprehensive two-dimensional liquid chromatography.

A method for choosing orthogonal columns for a specific sample set in on-line comprehensive two-dimensional liquid chromatography (LC×LC) was developed on the basis of the hydrophobic subtraction model. The method takes into account the properties of the sample analytes by estimating new F-weights for the prediction of orthogonality. We compared sets of F-weights and used these F-weights to predict orthogonal column combinations: (1) the standard F-weights determined by Gilroy et al. [1], (2) F-weights determined from the retention of sample analytes, and the same procedure of calculation as described by Gilroy et al. [1], (3) F-weights determined from the retention of sample analytes but using principal component analysis (PCA) for the estimation, and (4) the Gilroy F-weights modified by excluding the C-term in the hydrophobic subtraction model, as suggested by Dolan and Snyder [2]. The retention of 13 neutral and 4 acidic oxygenated polycyclic aromatic compounds (PACs) and 3 nitrogen-containing PAC bases was measured isocratically on 12 columns. The isocratic runs were used to determine the hydrophobic subtraction model analyte parameters, and these were used to estimate new F-weights and predict orthogonal column combinations. LC×LC-DAD analysis was then performed on a test mix using these column sets. We found that the column combination predicted from the new F-weights provide a more orthogonal separation of the PACs than those predicted using the standard F-weights and the F-weights modified by excluding the C-term. This emphasizes the necessity of considering the nature of the sample when choosing orthogonal columns.

New resistance-correlated saponins from the insect-resistant crucifer Barbarea vulgaris.

Isolation and characterization of plant constituents responsible for insect resistance are of the utmost importance for better understanding of insect-host plant interactions, for selection and breeding of resistant plant varieties, and for development of natural insecticides to be used in future sustainable agriculture and food production. In this study, 3-O-cellobiosyl-cochalic acid (1), 3-O-cellobiosyl-gypsogenin (3), and 3-O-cellobiosyl-4-epihederagenin (4) were isolated from the glabrous type of Barbarea vulgaris var. arcuata exhibiting resistance to the flea beetle Phyllotreta nemorum. In addition to the new constituents, 3-O-cellobiosyl-hederagenin (2), a known insect repellant, was identified. The structures were established by one- and/or two-dimensional homo- and heteronuclear NMR experiments acquired at 800 MHz and by fragmentation and high-resolution mass spectrometric analysis. Compounds 1, 3, and 4 are glycosides of cochalic acid, gypsogenin, and 4-epihederagenin, respectively, none of which have previously been identified in Brassicaceae. Compounds 3 and 4 have both recently been targeted as unidentified constituents exhibiting correlation with P. nemorum resistance, but this is the first report of their structures.

Potato glycoalkaloids in soil-optimising liquid chromatography-time-of-flight mass spectrometry for quantitative studies.

Potato glycoalkaloids are produced in high amounts in potato fields during the growth season and losses to soil potentially impact shallow groundwater and via tiles to fresh water ecosystems. A quantitative liquid chromatography-electrospray ionization time-of-flight mass spectrometry (LC-ESI-TOF-MS) method for determination and quantification of potato glycoalkaloids and their metabolites in aqueous soil extracts was developed. The LC-ESI-TOF-MS method had linearities up to 2000microg/L for alpha-solanine and alpha-chaconine and up to 760microg/L for solanidine. No matrix effect was observed, and the detection limits found were in the range 2.2-4.7microg/L. The method enabled quantification of the potato glycoalkaloids in environmental samples.

The biosynthetic pathway for aurofusarin in Fusarium graminearum reveals a close link between the naphthoquinones and naphthopyrones.

Fungal polyketide biosynthesis typically involves multiple enzymatic steps and the encoding genes are often found in gene clusters. A gene cluster containing PKS12, the polyketide synthase gene responsible for the synthesis of the pigment aurofusarin, was analysed by gene replacement using Agrobacterium tumefaciens-mediated transformation to determine the biosynthesis pathway of aurofusarin. Replacement of aurR1 with hygB shows that it encodes a positively acting transcription factor that is required for the full expression of PKS12, aurJ, aurF, gip1 and FG02329.1, which belong to the gene cluster. AurR1 and PKS12 deletion mutants are unable to produce aurofusarin and rubrofusarin. Bio- and chemoinformatics combined with chemical analysis of replacement mutants (DeltaaurJ, DeltaaurF, Deltagip1, DeltaaurO and DeltaPKS12) indicate a five-step enzyme catalysed pathway for the biosynthesis of aurofusarin, with rubrofusarin as an intermediate. This links the biosynthesis of naphthopyrones and naphthoquinones together. Replacement of the putative transcription factor aurR2 results in an increased level of rubrofusarin relative to aurofusarin. Gip1, a putative laccase, is proposed to be responsible for the dimerization of two oxidized rubrofusarin molecules resulting in the formation of aurofusarin.