Benefits of Ion Mobility Separation in GC-APCI-HRMS Screening: From the Construction of a CCS Library to the Application to Real-World Samples.

The performance of gas chromatography (GC) combined with the improved identification properties of ion mobility separation coupled to high-resolution mass spectrometry (IMS-HRMS) is presented as a promising approach for the monitoring of (semi)volatile compounds in complex matrices. The soft ionization promoted by an atmospheric pressure chemical ionization (APCI) source designed for GC preserves the molecular and/or quasi-molecular ion information enabling a rapid, sensitive, and efficient wide-scope screening. Additionally, ion mobility separation (IMS) separates species of interest from coeluting matrix interferences and/or resolves isomers based on their charge, shape, and size, making IMS-derived collision cross section (CCS) a robust and matrix-independent parameter comparable between instruments. In this way, GC-APCI-IMS-HRMS becomes a powerful approach for both target and suspect screening due to the improvements in (tentative) identifications. In this work, mobility data for 264 relevant multiclass organic pollutants in environmental and food-safety fields were collected by coupling GC-APCI with IMS-HRMS, generating CCS information for molecular ion and/or protonated molecules and some in-source fragments. The identification power of GC-APCI-IMS-HRMS for the studied compounds was assessed in complex-matrix samples, including fish feed extracts, surface waters, and different fruit and vegetable samples.

Identification of new, very long-chain polyunsaturated fatty acids in fish by gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with atmospheric pressure chemical ionization.

The characterization of very long-chain (>C24) polyunsaturated fatty acids (VLC-PUFAs), which are essential in the vision, neural function, and reproduction of vertebrates, is challenging because of the lack of reference standards and their very low concentrations in certain lipid classes. In this research, we have developed a new methodology for VLC-PUFA identification based on gas chromatography coupled to quadrupole/time-of-flight mass spectrometry with an atmospheric pressure chemical ionization source (GC-APCI-QTOF MS). The mass accuracy attainable with the innovative QTOF instrument, together with the soft ionization of the APCI source, provides valuable information on the intact molecule, traditionally lost with electron ionization sources due to the extensive fragmentation suffered. We have identified, for the first time, VLC-PUFAs with chains up to 44 carbons in eyes, brain, and gonads of gilthead sea bream, a commercially important fish in the Mediterranean. The added value of ion mobility-mass spectrometry (IMS), recently developed in combination with GC-QTOF MS, and the contribution of the collisional cross section (CCS) parameter in the characterization of novel VLC-PUFAs (for which reference standards are not available) have been also evaluated. The methodology developed has allowed assessing qualitative differences between farmed and wild fish, and opens new perspectives in a still scarcely known field of research.

LC-MS/MS method for the determination of organophosphorus pesticides and their metabolites in salmon and zebrafish fed with plant-based feed ingredients.

The composition of Atlantic salmon feed has changed considerably over the last two decades from being marine-based (fishmeal and fish oil) to mainly containing plant ingredients. Consequently, concern related to traditional persistent contaminants typically associated with fish-based feed has been replaced by other potential contaminants not previously associated with salmon farming. This is the case for many pesticides, which are used worldwide to increase food production, and may be present in plant ingredients. Earlier studies have identified two organophosphorus pesticides, chlorpyrifos-methyl and pirimiphos-methyl, in plant ingredients used for aquafeed production. In the present study, we developed a reliable and sensitive analytical method, based on liquid chromatography coupled to tandem mass spectrometry, for the determination of these pesticides and their main metabolites in warm water (zebrafish) and cold water (Atlantic salmon) species, where possible differences in metabolites could be expected. The method was tested in whole zebrafish and in different salmon tissues, such as muscle, bile, kidney, fat, and liver. The final objective of this work was to assess kinetics of chlorpyrifos-methyl and pirimiphos-methyl and their main metabolites in fish tissue, in order to fill the knowledge gaps on these metabolites in fish tissues when fed over prolonged time.

Simultaneous determination of dechloranes, polybrominated diphenyl ethers and novel brominated flame retardants in food and serum.

A sensitive method for the simultaneous quantification of dechloranes, polybrominated diphenyl ethers (PBDEs) and novel brominated flame retardants (NBFRs) has been developed for gas chromatography (GC) coupled to tandem mass spectrometry operating in electron capture negative ionization (ECNI) mode. The major advance has been achieved by combining selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes in well-defined time windows, to determine dechloranes, PBDEs and NBFRs at picogram per gram level in one single analysis in complex matrix biological samples. From the chromatographic point of view, efforts were devoted to study several injection modes using multimode inlet (MMI) in order to obtain low instrumental detection limits, necessary for trace compounds such as Dechlorane Plus (DP) isomers. Method performance was also evaluated: calibration curves were linear from 20 fg µL-1 to 100 pg µL-1 for the studied compounds, with method detection limits at levels of 50 fg g-1 for DPs. Repeatability and reproducibility, expressed as relative standard deviation, were better than 5% even in solvent vent mode for the injection of standards. The application to a wide range of complex samples (including food, human and animal serum samples) indicated a sensitive and reliable way to quantify at the picogram per gram level 4 halogenated norbornenes (HNs), Dechlorane Plus (anti-DP and syn-DP) and 2 of their homologues (Dechlorane-602 and Dechlorane-603), 11 PBDE congeners (no. 28, 47, 49, 66, 85, 99, 100, 153, 154, 183 and 209) and 5 novel BFRs, i.e. decabromodiphenyl ethane (DBDPE), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), hexabromobenzene (HBB), 2,3,4,5-tetrabromo-ethylhexyl-benzoate (TBB) and tetrabromophthalate (TBPH). Graphical Abstract GC-ECNI-MS/MS chromatograms showing the most sensitive transition for DPs when injecting 2 µL of a 16 fg/µL standard solution of s-DP and a-DP at three different source temperatures.

Potential of gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry for screening and quantification of hexabromocyclododecane.

A fast method for the screening and quantification of hexabromocyclododecane (sum of all isomers) by gas chromatography using a triple quadrupole mass spectrometer with atmospheric pressure chemical ionization (GC-APCI-QqQ) is proposed. This novel procedure makes use of the soft atmospheric pressure chemical ionization source, which results in less fragmentation of the analyte than by conventional electron impact (EI) and chemical ionization (CI) sources, favoring the formation of the [M - Br](+) ion and, thus, enhancing sensitivity and selectivity. Detection was based on the consecutive loses of HBr from the [M - Br](+) ion to form the specific [M - H5Br6](+) and [M - H4Br5](+) ions, which were selected as quantitation (Q) and qualification (q) transitions, respectively. Parameters affecting ionization and MS/MS detection were studied. Method performance was also evaluated; calibration curves were found linear from 1 pg/µL to 100 pg/µL for the total HBCD concentration; instrumental detection limit was estimated to be 0.10 pg/µL; repeatability and reproducibility, expressed as relative standard deviation, were better than 7% in both cases. The application to different real samples [polyurethane foam disks (PUFs), food, and marine samples] pointed out a rapid way to identify and allow quantification of this compound together with a number of polybrominated diphenyl ethers (BDE congeners 28, 47, 66, 85, 99, 100, 153, 154, 183, 184, 191, 196, 197, and 209) and two other novel brominated flame retardants [i.e., decabromodiphenyl ethane (DBDPE) and 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE)] because of their presence in the same fraction when performing the usual sample treatment.

Novel analytical approach for brominated flame retardants based on the use of gas chromatography-atmospheric pressure chemical ionization-tandem mass spectrometry with emphasis in highly brominated congeners.

The analysis of brominated flame retardants (BFRs) commonly relies on the use of gas chromatography coupled to mass spectrometry (GC-MS) operating in electron ionization (EI) and electron capture negative ionization (ECNI) modes using quadrupole, triple quadrupole, ion trap, and magnetic sector analyzers. However, these brominated contaminants are examples of compounds for which a soft and robust ionization technique might be favorable since they show high fragmentation in EI and low specificity in ECNI. In addition, the low limits of quantification (0.01 ng/g) required by European Commission Recommendation 2014/118/EU on the monitoring of traces of BFRs in food put stress on the use of highly sensitive techniques/methods. In this work, a new approach for the extremely sensitive determination of BFRs taking profit of the potential of atmospheric pressure chemical ionization (APCI) combined with GC and triple quadrupole (QqQ) mass analyzer is proposed. The objective was to explore the potential of this approach for the BFRs determination in samples at pg/g levels, taking marine samples and a cream sample as a model. Ionization and fragmentation behavior of 14 PBDEs (congeners 28, 47, 66, 85, 99, 100, 153, 154, 183, 184, 191, 196, 197, and 209) and two novel BFRs, decabromodiphenyl ethane (DBDPE) and 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE), in the GC-APCI-MS system has been investigated. The formation of highly abundant (quasi) molecular ion was the main advantage observed in relation to EI. Thus, a notable improvement in sensitivity and specificity was observed when using it as precursor ion in tandem MS. The improved detectability (LODs < 10 fg) achieved when using APCI compared to EI has been demonstrated, which is especially relevant for highly brominated congeners. Analysis of samples from an intercomparison exercise and samples from the marine field showed the potential of this approach for the reliable identification and quantification at very low concentration levels.

Atmospheric-pressure chemical ionization tandem mass spectrometry (APGC/MS/MS) an alternative to high-resolution mass spectrometry (HRGC/HRMS) for the determination of dioxins.

The use of a new atmospheric-pressure chemical ionization source for gas chromatography (APGC) coupled with a tandem quadrupole mass spectrometry (MS/MS) system, as an alternative to high-resolution mass spectrometry (HRMS), for the determination of PCDDs/PCDFs is described. The potential of using atmospheric-pressure chemical ionization (APCI) coupled to a tandem quadrupole analyzer has been validated for the identification and quantification of dioxins and furans in different complex matrices. The main advantage of using the APCI source is the soft ionization at atmospheric pressure, which results in very limited fragmentation. APCI mass spectra are dominated by the molecular ion cluster, in contrast with the high energy ionization process under electron ionization (EI). The use of the molecular ion as the precursor ion in MS/MS enhances selectivity and, consequently, sensitivity by increasing the signal-to-noise ratios (S/N). For standard solutions of 2,3,7,8-TCDD, injections of 10 fg in the splitless mode on 30- or 60-m-length, 0.25 mm inner diameter (id), and 25 µm film thickness low-polarity capillary columns (DB5MS type), signal-to-noise (S/N) ratios of >10:1 were routinely obtained. Linearity was achieved in the region (correlation coefficient of r(2) > 0.998) for calibration curves ranging from 100 fg/µL to 1000 pg/µL. The results from a wide variety of complex samples, including certified and standard reference materials and samples from several QA/QC studies, which were previously analyzed by EI HRGC/HRMS, were compared with the results from the APGC/MS/MS system. Results between instruments showed good agreement both in individual congeners and toxic equivalence factors (TEQs). The data show that the use of APGC in combination with MS/MS for the analysis of dioxins has the same potential, in terms of sensitivity and selectivity, as the traditional HRMS instrumentation used for this analysis. However, the APCI/MS/MS system, as a benchtop system, is much easier to use.

Non-target screening with high-resolution mass spectrometry: critical review using a collaborative trial on water analysis.

In this article, a dataset from a collaborative non-target screening trial organised by the NORMAN Association is used to review the state-of-the-art and discuss future perspectives of non-target screening using high-resolution mass spectrometry in water analysis. A total of 18 institutes from 12 European countries analysed an extract of the same water sample collected from the River Danube with either one or both of liquid and gas chromatography coupled with mass spectrometry detection. This article focuses mainly on the use of high resolution screening techniques with target, suspect, and non-target workflows to identify substances in environmental samples. Specific examples are given to emphasise major challenges including isobaric and co-eluting substances, dependence on target and suspect lists, formula assignment, the use of retention information, and the confidence of identification. Approaches and methods applicable to unit resolution data are also discussed. Although most substances were identified using high resolution data with target and suspect-screening approaches, some participants proposed tentative non-target identifications. This comprehensive dataset revealed that non-target analytical techniques are already substantially harmonised between the participants, but the data processing remains time-consuming. Although the objective of a "fully-automated identification workflow" remains elusive in the short term, important steps in this direction have been taken, exemplified by the growing popularity of suspect screening approaches. Major recommendations to improve non-target screening include better integration and connection of desired features into software packages, the exchange of target and suspect lists, and the contribution of more spectra from standard substances into (openly accessible) databases. Graphical Abstract Matrix of identification approach versus identification confidence.

Untargeted metabolomics unravels distinct gut microbial metabolites derived from plant-based and animal-origin proteins using in vitro modeling.

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.

Identification and quantification of flavor compounds in smoked tuna fish based on GC-Orbitrap volatolomics approach.

Cold smoking enhances the appeal of fish products, offering consumers a smooth texture and a delicate smoky flavor. This study aims to explore variations in the volatile profile from different exposure times during cold smoking processing (light, moderate, and full-cure) in tune samples. An innovative untargeted analytical approach, headspace solid-phase microextraction combined with gas chromatography and a hybrid quadrupole-orbitrap mass analyzer, was employed to identify 86 volatiles associated with the cold smoking process. Most of these compounds, including phenols, furan derivates, aldehydes, cyclic ketones, and different aromatic species, were found to contribute to the smoke odor. The development of a QuEChERS-based extraction and clean-up method facilitated the quantification of 25 relevant smoky markers across all smoking degrees, revealing significant concentration differences after 15 h of smoking. This research sheds light on the dynamics of cold smoking impact and its on the flavor profile and safety quality of processed fish products.

High resolution mass spectrometry-based screening for the comprehensive investigation of organic micropollutants in surface water and wastewater from Pasto city, Colombian Andean highlands.

The complexity of the aquatic environment scenario, including the impact of urban wastewater, together with the huge number of potential hazardous compounds that may be present in waters, makes the comprehensive characterization of the samples an analytical challenge, particularly in relation to the presence of organic micropollutants (OMPs). Nowadays, the potential of high-resolution mass spectrometry (HRMS) for wide-scope screening in environmental samples is out of question. Considering the physicochemical characteristics of OMPs, the coupling of liquid (LC) and gas chromatography (GC) to HRMS is mandatory. In this work, we have explored the combined use of LC and GC coupled to Quadrupole-Time-of-Flight Mass Spectrometry (QTOF MS) for screening of surface water and wastewater samples from Pasto (Nariño), a town of the Colombian Andean highlands (average altitude 2527 m), located in an important agricultural area. The upper basin of the Pasto River is impacted by phytosanitary products used in different crops, whereas the domestic wastewater is directly discharged into the river without any treatment, enhancing the anthropogenic impact on the water quality. The OMP searching was made by target (standards available) and suspect (without standards) approaches, using home-made databases containing >2000 compounds. Up to 15 pesticides (7 insecticides, 6 fungicides and 2 herbicides) were identified in the sampling point of the Pasto River up to the town, while no pharmaceuticals were found at this site, illustrating the impact of agriculture practices. On the contrary, 14 pharmaceuticals (7 antibiotics and 3 analgesics, among others) were found in river samples collected in the middle and down to the town sites, revealing the impact of the urban population. Interestingly, some transformation products, including metabolites, such as carbofuran-3-hydroxy and 4-acetylamino antipyrine were identified in the screening. Based on these data, future monitoring will apply target quantitative LC-MS/MS methods for the most relevant compounds identified.

Discovery of Food Intake Biomarkers Using Metabolomics.

Due to the high impact of diet exposure on health, it is crucial the generation of robust data of regular dietary intake, hence improving the accuracy of dietary assessment. The metabolites derived from individual food or group of food have great potential to become biomarkers of food intake (BFIs) and provide more objective food consumption measurements.Herein, it is presented an untargeted metabolomic workflow for the discovery BFIs in blood and urine samples, from the study design to the biomarker identification. Samples are analyzed by liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS). A wide variety of compounds are covered by separate analyses of medium to nonpolar molecules and polar metabolites based on two LC separations as well as both positive and negative electrospray ionization. The main steps of data treatment of the comprehensive data sets and statistical analysis are described, as well as the principal considerations for the BFI identification.

Untargeted metabolomics approach using UHPLC-IMS-QTOF MS for surface body samples to identify low-volatility chemosignals related to maternal care in mice.

The present study is focused on the determination of low-volatile chemosignals excreted or secreted by mouse pups in their early days of life involved in maternal care induction in mice adult females. Untargeted metabolomics was employed to differentiate between samples collected with swabs from facial and anogenital area from neonatal mouse pups receiving maternal care (first two weeks of life) and the elder mouse pups in the weaning period (4th week old). The sample extracts were analysed by ultra-high pressure liquid chromatography (UHPLC) coupled to ion mobility separation (IMS) in combination with high resolution mass spectrometry (HRMS). After data processing with Progenesis QI and multivariate statistical analysis, five markers present in the first two weeks of mouse pups life and putatively involved in materno-filial chemical communication were tentatively identified: arginine, urocanic acid, erythro-sphingosine (d17:1), sphingosine (d18:1) and sphinganine. The four-dimensional data and the tools associated to the additional structural descriptor obtained by IMS separation were of great help in the compound identification. The results demonstrated the great potential of UHPLC-IMS-HRMS based untargeted metabolomics to identity putative pheromones in mammals.

LC-IMS-HRMS for identification of biomarkers in untargeted metabolomics: The effects of pterostilbene and resveratrol consumption in liver steatosis, animal model.

Untargeted metabolomics with the combination of ion mobility separation coupled to high resolution mass spectrometry (IMS-HRMS) was applied to investigate the impact of resveratrol and pterostilbene supplementation on the metabolic fingerprint of the Wistar rats liver with induced liver steatosis. RP-LC and HILIC in both ionisation modes were employed to analyse the liver samples (n = 40) from Wistar rats fed with a high-fat and high-fructose diet, supplemented or not with resveratrol and pterostilbene. After univariate and multivariate statistical analysis, 34 metabolites were highlighted in the different diets and elucidated. Despite the structural similarity, different alterations in liver metabolism were observed by the supplementations. Resveratrol treatment was characterised by the alteration in metabolism of 17 lysophospholipids, while pterostilbene affected some vitamins and derivatives, among others. IMS has demonstrated great potential in the elucidation process thanks to the additional structural descriptor the CCS (Å2), providing more confidence in the identification.

A comprehensive study on diesel oil bioremediation under microcosm conditions using a combined microbiological, enzymatic, mass spectrometry, and metabarcoding approach.

This study aims at the application of a marine fungal consortium (Aspergillus sclerotiorum CRM 348 and Cryptococcus laurentii CRM 707) for the bioremediation of diesel oil-contaminated soil under microcosm conditions. The impact of biostimulation (BS) and/or bioaugmentation (BA) treatments on diesel-oil biodegradation, soil quality, and the structure of the microbial community were studied. The use of the fungal consortium together with nutrients (BA/BS) resulted in a TPH (Total Petroleum Hydrocarbon) degradation 42% higher than that obtained by natural attenuation (NA) within 120 days. For the same period, a 72 to 92% removal of short-chain alkanes (C12 to C19) was obtained by BA/BS, while only 3 to 65% removal was achieved by NA. BA/BS also showed high degradation efficiency of long-chain alkanes (C20 to C24) at 120 days, reaching 90 and 92% of degradation of icosane and heneicosane, respectively. In contrast, an increase in the levels of cyclosiloxanes (characterized as bacterial bioemulsifiers and biosurfactants) was observed in the soil treated by the consortium. Conversely, the NA presented a maximum of 37% of degradation of these alkane fractions. The 5-ringed PAH benzo(a)pyrene, was removed significantly better with the BA/BS treatment than with the NA (48 vs. 38 % of biodegradation, respectively). Metabarcoding analysis revealed that BA/BS caused a decrease in the soil microbial diversity with a concomitant increase in the abundance of specific microbial groups, including hydrocarbon-degrading (bacteria and fungi) and also an enhancement in soil microbial activity. Our results highlight the great potential of this consortium for soil treatment after diesel spills, as well as the relevance of the massive sequencing, enzymatic, microbiological and GC-HRMS analyses for a better understanding of diesel bioremediation.

Multiclass determination of 66 organic micropollutants in environmental water samples by fast gas chromatography-mass spectrometry.

A multiresidue method has been developed for quantification and identification of 66 multiclass priority organic pollutants in water by fast gas chromatography (GC) coupled to mass spectrometry (MS). Capabilities and limitations of single quadrupole mass spectrometer as detector in fast GC were studied evaluating the chromatographic responses in terms of sensitivity and chromatographic peak shapes, as they were influenced by scan time. The number of monitored ions in a selected ion monitoring (SIM) group strongly conditioned the scan time and subsequently the number of data points per peak. A compromise between peak shape and scan time was adopted in order to reach the proper conditions for quantitative analysis. An average of 10-15 points per peak was attained for most compounds, involving scan times between 0.1 and 0.22 s. The method was validated for mineral, surface, and groundwater. A solid-phase extraction pre-concentration step using C(18) cartridges was applied. Four isotopically labeled standards were added to the samples before extraction and used as surrogates to ensure a reliable quantification. Analyses were performed by GC-MS in electron ionization mode, monitoring the three most abundant and/or specific ions for each compound and using the intensity ratios as a confirmatory parameter. With a chromatographic run of less than 10 min, SIM mode provided excellent sensitivity and identification capability due to the monitoring of three ions and the evaluation of their intensity ratio. Limits of detection below 10 ng/L were reached for most of the 66 compounds in the three matrices studied. Accuracy and precision of the method were evaluated by means of recovery experiments at two fortification levels (10 and 100 ng/L), obtaining recoveries between 70% and 120% in most cases and relative standard deviations below 20%. The possibilities of a simultaneous SIM scan method have also been explored for non-target qualitative analysis. The developed method has been applied to the analysis of surface water samples collected from the Mediterranean region of Spain.

Determination of very long-chain polyunsaturated fatty acids from 24 to 44 carbons in eye, brain and gonads of wild and cultured gilthead sea bream (Sparus aurata).

Very long-chain (> C24) polyunsaturated fatty acids (VLC-PUFA) play an important role in the development of nervous system, retinal function and reproductive processes in vertebrates. Their presence in very small amounts in specific lipid classes, the lack of reference standards and their late elution in chromatographic analyses render their identification and, most important, their quantification, still a challenge. Consequently, a sensitive and feasible analytical methodology is needed. In this work, we have studied the effect of chain length, as well as the number and position of unsaturations (or double bonds) on the response of GC-APCI-(Q)TOF MS, to establish an analytical method for VLC-PUFA quantification. The developed methodology allows the quantification of these compounds down to 2.5 × 10-3 pmol/mg lipid. The reduction of VLC-PUFA levels in lipid fractions of the organs from the herein sampled farmed fish suggesting a yet undetected effect on these compounds of high vegetable oil aquafeed formulations, that currently dominate the market.

Use of soft and hard ionization techniques for elucidation of unknown compounds by gas chromatography/time-of-flight mass spectrometry.

Investigation of trace-level non-target compounds by gas chromatography/mass spectrometry (GC/MS) often is a challenging task that requires powerful software tools to detect the unknown components, to obtain the deconvoluted mass spectra, and to interpret the data if no acceptable library match is obtained. In this paper, the complementary use of electron ionization (EI) and chemical ionization (CI) is investigated in combination with GC/time-of-flight (TOF) MS for the elucidation of organic non-target (micro)contaminants in water samples. Based on accurate mass measurement of the molecular and fragment ions from the TOF MS, empirical formulae were calculated. Isotopic patterns, carbon number prediction filter and nitrogen rule were used to reduce the number of possible formulae. The candidate formulae were searched in databases to find possible chemical structures. Selection from possible structure candidates was achieved using information on substructures and observed neutral losses derived from the fragment ions. Four typical examples (bifenazate, boscalid, epoxiconazole, and fenhexamid) are used to illustrate the methodology applied and the various difficulties encountered in this process. Our results indicate that elucidation of unknowns cannot be achieved by following a standardized procedure, as both expertise and creativity are necessary in the process.

Novel sampling strategy for alive animal volatolome extraction combined with GC-MS based untargeted metabolomics: Identifying mouse pup pheromones.

In this study, we identify 11 mouse pup volatiles putatively involved in maternal care induction in adult females. For this purpose, we have adapted the dynamic headspace methodology to extract the volatolome of whole alive animals. Untargeted metabolomic methodology was used to compare the volatolome of neonatal (4-6 days) with elder pups until the age of weaning (21-23 days old). Pup volatolome was analyzed by gas chromatography (GC) coupled to single quadrupole mass spectrometry (MS) using automated thermal desorption for sample introduction. After data processing and multivariate statistical analysis, comparison with NIST spectral library allowed identifying compounds secreted preferentially by neonatal pups: di(propylen glycol) methyl ether, 4-nonenal, di(ethylene glycol) monobutyl ether, 2-phenoxyethanol, isomethyl ionone, tridecanal, 1,3-diethylbenzene, 1,2,4,5-tetramethylbenzene, 2-ethyl-p-xylene and tri(propylene glycol) methyl ether. Palmitic acid was enriched in the volatolome of fourth week youngsters compared to neonatal pups. The results demonstrated the great potential of the new sampling procedure combined with GC-MS based untargeted volatolomics to identify volatile pheromones in mammals.

Ecological risk assessment of pesticides in the Mijares River (eastern Spain) impacted by citrus production using wide-scope screening and target quantitative analysis.

The widespread use of pesticides, especially in agricultural areas, makes necessary to control their presence in surrounding surface waters. The current study was designed to investigate the occurrence and ecological risks of pesticides and their transformation products in a Mediterranean river basin impacted by citrus agricultural production. Nineteen sites were monitored in three campaigns distributed over three different seasons. After a qualitative screening, 24 compounds was selected for subsequent quantitative analysis. As expected, the lower section of the river was most contaminated, with total concentration >5 µg/L in two sites near to the discharge area of wastewater treatment plants. The highest concentrations were found in September, after agricultural applications and when the river flow is reduced. Ecological risks were calculated using two mixture toxicity approaches (Toxic Unit and multi-substance Potentially Affected Fraction), which revealed high acute and chronic risks of imidacloprid to invertebrates, moderate-to-high risks of diuron, simazine and 2,4-D for primary producers, and moderate-to-high risks of thiabendazole for invertebrates and fish. This study shows that intensive agricultural production and the discharge of wastewater effluents containing pesticide residues from post-harvest citrus processing plants are threatening freshwater biodiversity. Further actions are recommended to control pesticide use and to reduce emissions.