Non-targeted Screening Approach Research Articles

Nontargeted Screening for the Verification of Allergenic Ingredients and Perfume Authenticity by GC-ecTOF-MS

Fraudulent products are ubiquitous in all markets. Besides the financial aspect, a major issue regarding products adulteration for society and the environment is the missing regulation and control of these goods. Therefore, harmful and toxic compounds in fraudulent products may become a risk for human health and the environment. Even small amounts of toxic substances can still have damaging effects. Thus, a sensitive and reliable identification of compounds is needed. Novel technologies are necessary to use identifying and preventing fraud and related risks. Goods from the food, flavor, and fragrance markets often contain volatile organic compounds (VOCs), which include most allergenic fragrances. For the detection and identification of these substances, gas chromatographic separation hyphenated with high resolution mass spectrometry (GC–HRMS) is an ideal instrumental technique. GC–simultaneous electron and chemical ionization (ec) time-of-flight (TOF)-MS generates various types of information via simultaneous ec–HRMS. Advantages are given for target, known unknown, and unknown unknown data analysis by generating various types of ions within one single experimental GC–MS run. In this study, the experimental nontargeted screening approach and corresponding data analysis workflows—simultaneously using molecular ion information and structural information—are presented for the molecular identification and authenticity verification process from a brand perfume using GC–ecTOF-MS.

Finding features - variable extraction strategies for dimensionality reduction and marker compounds identification in GC-IMS data

Gas chromatography hyphenated to ion mobility spectrometry (GC-IMS) is a powerful, two-dimensional separation and detection technique for volatile organic compounds (VOC). Low detection limits, high selectivity and robust operation characterize it as an ideal tool for non-target screening (NTS) approaches. Combined with multivariate data analysis, it has been successfully applied to several areas in food science, such as authenticity control and flavor profiling. The recorded raw data feature high numbers of variables due to the high scan speeds of the instrument. Additionally, NTS approaches - by design - record more data than required. Therefore, reducing the number of variables is a key step in any machine learning pipeline to reduce overfitting, overlong training times and model complexity. The aim of the study is a comparison between the two most used dimensionality reduction techniques, PCA and PLS, regarding interpretability, as a tool to find marker compounds, and performance as a preprocessing step for supervised learning. Both feature per variable visualizations, which allows easy interpretation of results and retains a connection to the input data, which can lead to the discovery of marker compounds. A GC-IMS dataset about the botanical origin of honey is used, and all formatting steps necessary to apply PCA and PLS to higher dimensional data and obtain intuitive figures are explained. To evaluate effectiveness as a preprocessing step in a supervised pipeline four supervised algorithms were fitted with PCA or PLS variable reduction. PLS proved to be a more effective step in a supervised workflow in terms of accuracy, while PCA is highly effective for revealing preprocessing weaknesses such as misalignments.

Practical high resolution mass spectrometry screening approach and propositions for adequate identification criteria as well as quality assurance and validation specifics

The use of High Resolution Mass Spectrometry (HRMS) for comprehensive (non-targeted) screening approaches has increased over the past decade in forensic toxicology. To fully implement this technique in a routine laboratory with increasing demands concerning throughput and quality (e.g. ISO 17025), the method should be easy to apply and data interpretation should be straightforward. Few guidelines of the forensic field specifically addressed specific issues concerning identification criteria, adequate validation experiments and quality assurance (QA) for HRMS approaches. The aim of this study is to describe a practical HRMS approach and to propose suggestions for validation and QA. Sample preparation consisted of a protein precipitation in ice-cold acetonitrile before evaporation and reconstitution in ammonium formate buffer. ß-theophylline was added as internal standard (IS). Three μL of the extract were then injected onto a 150 mm × 2.1 mm HSST3 C18 UPLC column. The applied gradient started with 87% water/ammonium formate 5 mM pH3 (A) and 13% acetonitrile 0,1% formic acid (B). At 10 minutes, 50% A and B was linearly obtained at a constant flow rate of 0,4 mL/min. At 16 min., 1% A and 99% B was programmed to be reached exponentially (curve 7) and at 18,5 min. starting conditions were applied again up to the 21th minute. Data were acquired on a Xevo G2 XS-QTOF (Waters, Milford, US) and processed using the UNIFI toxicology screening solution, combined with the web-based HighResNPS database. Our solution is a 21 minutes run based on the Waters UNIFI Forensic Toxicology Screening Application with improvements to detect New Psychoactive Substances (NPS) such as synthetic cannabinoids (SCRAs) which originally eluted in the washing phase. About 15 samples in 5 hours could be analysed with simultaneous data processing and reporting. The automated workflow filters data on mass accuracy (< 5 ppm), fragment ion (min 1) and a response (>10,000). Additional information for visual inspection concern isotope match, retention time (observed and expected) and fragments (found and count) is given. QA is set-up via an initial suitability test looking into sensitivity, reproducibility, mass accuracy and resolution (full system information including LC, MS and contamination level control), quality control samples (mix, blanks) and proficiency tests (ACQ Science EEQ, GTFCh QSA). The method was validated for blood with regards to selectivity, sensitivity (LOI), matrix effects, stability and carry-over for 25 compounds eluting throughout the analytical run (3-Fluorophenmetrazine, 5F-ADB, Acryloyl fentanyl, Acryloyl fentanyl, Bromazepam, Deschloroetizolam, Diazepam, Diclazepam, Diphenidine, EG-018, Fentanyl, Flunitrazolam, Fluoxetine, FUB-144, Furanyfentanyl, Gabapentin, Ketamine, Mephedrone, MMB-022, NM-2AI, NSI-189, Oxazepam, Risperidone, Tramadol, Trazodone, Valeryl fentanyl). Sensitivity throughout the batch was normalized via the intensity of the IS to establish the robustness and potential impact on data interpretation. Selectivity was evaluated via blanks versus other samples in the same batch to evaluated ‘false positive’ hits in the database. In addition, authentic samples were applied to compare the HRMS TOF data with established LC-MS/MS methods. Discussion As more laboratories consider HRMS for comprehensive drugs screening, a consistent approach to data analysis and compound identification will be crucial. Carefully selecting, validating and controlling the positivity criteria used in HRMS data analysis is a step in this direction and necessary for ISO17025 accredited laboratories. The choices made in the proposed workflow are evaluated via authentic cases. Identification and validation criteria will be checked against current international guidelines. The developed targeted HRMS workflow improves the simplicity of the procedure and limits the time-constraints and the need of highly experienced personnel for interpretation.

FindPFΔS: Non-Target Screening for PFAS─Comprehensive Data Mining for MS2 Fragment Mass Differences.

The limited availability of analytical reference standards makes non-target screening approaches based on high-resolution mass spectrometry increasingly important for the efficient identification of unknown PFAS (per- and polyfluoroalkyl substances) and their TPs. We developed and optimized a vendor-independent open-source Python-based algorithm (FindPFΔS = FindPolyFluoroDeltas) to search for distinct fragment mass differences in MS/MS raw data (.ms2-files). Optimization with PFAS standards, two pre-characterized paper and soil samples (iterative data-dependent acquisition), revealed Δ(CF2)n, ΔHF, ΔCnH3F2n-3, ΔCnH2F2n-4, ΔCnHF2n-5, ΔCnF2nSO3, ΔCF3, and ΔCF2O as relevant and selective fragment differences depending on applied collision energies. In a PFAS standard mix, 94% (36 of 38 compounds from 10 compound classes) could be found by FindPFΔS. The use of fragment differences was applicable to a wide range of PFAS classes and appears as a promising new approach for PFAS identification. The influence of mass tolerance and intensity threshold on the identification efficiency and on the detection of false positives was systematically evaluated with the use of selected HR-MS2-spectra (20,998) from MassBank. To this end, with the use of FindPFΔS, we could identify different unknown PFAS homologues in the paper extracts. FindPFΔS is freely available as both Python source code on GitHub (https://github.com/JonZwe/FindPFAS) and as an executable windows application (https://doi.org/10.5281/zenodo.6797353) with a graphical user interface on Zenodo.

Arsenolipids in raw and cooked seafood products in southwest China: A non-targeted analysis

Arsenolipids are the primary form of arsenic in the fat of marine organisms. Because seafood is a common source of arsenic exposure and some arsenolipids are toxic, studying the abundance and species of arsenolipids in seafood is crucial for health risk assessment. Current arsenolipid research is confined by analytical techniques and limited to raw seafood analysis, despite the fact that most seafood is ingested cooked. Therefore, the aim of this study is to evaluate which seafood contributes to arsenolipid dietary intake and investigate the changes in arsenolipids before and after cooking. In Chongqing, China, popular seafood such as clam, shrimp, oyster, abalone, hairtail, and yellow croaker were collected. The raw and cooked samples prepared from these seafood products were examined using a non-targeted screening approach established for arsenolipids, which coupled high-performance liquid chromatography with data-independent high-resolution quadrupole-time-of-flight electrospray ionization tandem mass spectrometry. Arsenic-containing hydrocarbons (AsHC330, AsHC332, and AsHC360), arsenic-containing fatty acids (AsFA362, AsFA390, AsFA404, AsFA418, and AsFA422), trimethylarsine oxide, and thiolated trimethylarsinic acid were detected. The species of arsenolipids in each type of seafood remained intact after heating in the microwave oven. In cooked samples, the concentrations of AsFA362 and AsFA390 were significantly lower than in raw samples, whereas the concentrations of other arsenolipids were unchanged. Microwave cooking did not result in the thiolation of the detected arsenolipids. The most detected species in raw and cooked samples were AsFA362, AsFA390, and AsFA418. Most arsenolipid species were found in the highest levels in hairtails and yellow croakers. It is the first time that arsenolipids have been found in the oyster, abalone, abalone liver, and yellow croaker. The present study contributes to a better understanding of arsenolipids exposure from seafood, which is useful for assessing the health risks of arsenic.

Nontarget Approach to Identify Complexing Agents in Atmospheric Aerosol and Rainwater Samples

Atmospheric particles and droplets contain numerous organic substances, some of which form complexes with metal ions, significantly affecting bulk physicochemical properties and chemical reactivity. However, the detection and identification of complexing agents and their corresponding metal complexes remains an analytical challenge. In this study, we developed an LC/HRMS nontarget screening (NTS) approach which allows the selective detection of complexing agents in aerosol particle extracts and rainwater. To achieve this, a T-junction is installed between the LC outlet and the ion source, and a FeCl3 solution is added for postcolumn complexation. The resulting mass spectra are screened for the three characteristic iron(III)-complexes [M - H + FeCl3]-, [M - 2H + FeCl2]-, and [M - 3H + FeCl]- with mass differences (Δm/z) between the complexing agent and the iron complex of 160.8416, 124.8648, and 89.8959, respectively. Up to 29 di- or tricarboxylic acids were identified as complexing agents in aerosol particle samples from two different sites (Melpitz, Germany, and Wangdu, China) at concentrations as low as 50 nM. Thirteen complexing agents were detected even in measurements without postcolumn iron addition from complexation with background Fe3+ traces from the analytical system. At least for the highest concentrated complexing agents, the proposed screening approach can thus be exploited in a NTS approach without any device modification. Besides carboxylic acids, 4-nitrophenol and 4-nitrocatechol were identified as further complexing agents, demonstrating the applicability of the approach to other matrices and to a range of different complexing agents.

Occurrence and contamination profile of legacy and emerging per- and polyfluoroalkyl substances (PFAS) in Belgian wastewater using target, suspect and non-target screening approaches

With the growing concern regarding the health risks of per- and polyfluoroalkyl substances (PFAS), there is an increasing demand for the identification of emerging PFAS. This study provides a comprehensive investigation of legacy and emerging PFAS in 16 wastewater treatment plants (WWTPs) in Belgium using target, suspect, and non-target screening methods. Perfluorobutanoic acid (PFBA) and perfluoropentanoic acid (PFPeA) were the dominant compounds in most locations, whereas perfluorooctanoic acid (PFOA) was the most predominant PFAS in WWTP Deurne (Antwerp region). Using a suspect screening approach, 14 PFAS were annotated as confidence level (CL) of 4 or higher and 4 PFAS were annotated as CL 2a and 2b, including aqueous film forming foam (AFFF)-derived PFAS. The compound group of n:3 unsaturated fluorotelomer carboxylic acid was found using non-target screening in the wastewater from WWTP Deurne. Population exposure in a catchment area estimated using population-normalized mass loads (PNML) showed the highest value in the catchment area of WWTP Deurne, implying a potentially higher exposure to PFAS in this community.

Recognition and Health Impacts of Organic Pollutants with Significantly Different Proportions in the Gas Phase and Size-Fractionated Particulate Phase in Ambient Air.

The distributions of organic pollutants in the gas phase and size-fractionated particle phases can largely affect human health risks posed by them. Gas-particle partitioning and particle-size distributions of some known pollutants have been investigated. However, the pollutants which are more likely to enter the human body and cause strong adverse effects may be neglected. In this study, a nontargeted screening approach combining comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry and chemometrics was developed. Eighty-eight compounds with markedly different proportions in the gas phase and PM1, as well as 50 contaminants with significant differences in PM1 and particles with diameters of 1-2.5 μm, were identified. Of these compounds, 18 were found in the air for the first time. There were obvious discrepancies between the measured and predicted gas-particle partitioning coefficients for some pollutants, suggesting unexpected environmental fates and health risks. The human daily intakes through inhalation and dermal exposure to these pollutants were estimated with the International Commission on Radiological Protection deposition model and transdermal permeability model. A risk-based prioritization was performed. The results indicated that adverse effects posed by 9H-fluoren-9-one, 2-ethylhexyl 3-(3,5-di-tert-butyl-4-hydroxyphenyl) propionate, p-cumenol, 2,4-diisocyanato-1-methyl-benzene, bis(2-ethylhexyl) phthalate, perylene, (E)-cinnamaldehyde, 4-methyl-2-nitro-phenol, benzoic acid, and bis(2-methylpropyl) ester hexanedioic acid in ambient air may be more severe than those posed by conventionally monitored pollutants. The findings would facilitate raising concerns about these pollutants before they cause further severe and widespread impacts.

Modern Nontargeted Screening (NTS) Methods for Environmental Analysis

LCGC Europe spoke to Selina Tisler and Jan H. Christensen from the University of Copenhagen in Denmark, about recent research projects focusing on nontargeted screening (NTS) approaches for important environmental monitoring applications, including the analysis of chemical leaching from plastic bottles using liquid chromatography tandem mass spectrometry (LC–MS/MS) and sediment analysis using comprehensive two-dimensional gas chromatography–high-resolution MS (GC×GC–HRMS).

Long-term degradation of toluene and phenol in soil: Identification of transformation products and pathways via HRMS-based suspect and non-target screening

In this study, the long-term fate of toluene and phenol in the soil was investigated, and the transformation products (TPs) and pathways of these compounds were studied by a high resolution mass spectrometry (HRMS)-based suspect and non-target screening approach for the first time, and 9 and 12 transformation products were identified for toluene and phenol, respectively in the lab-exposed soil samples. Salicylaldehyde, 4-hydroxybenzaldehyde, and benzaldehyde were identified in toluene-contaminated field soil samples for the first time, and the main mechanisms involved in the biodegradation and detoxification of toluene and phenol in soil were oxidation, carboxylation, dehydroxylation, and ring fission amongst others. 2-oxoglutarate, TP165-A, TP165-B, TP172, and TP195 were identified as novel phenol transformation products, while salicylaldehyde, 2-oxoglutarate, TP165-A, and TP165-B were identified as novel toluene transformation products, providing new possible evidence for additional degradation pathways, which could give new insights into the fate of toluene and phenol during the natural attenuation process in the environment. Finally, salicylaldehyde, 4-OH-benzaldehyde, and 4-OH-benzoic acid which were detected at Level 1 identification confidence were suggested as indicator chemicals of toluene and phenol exposure in the contaminated field.

Occurrence and Contamination Profile of Legacy and Emerging Per- and Polyfluoroalkyl Substances (Pfas) in Belgian Wastewater Using Target, Suspect and Non-Target Screening Approaches

Occurrence and Contamination Profile of Legacy and Emerging Per- and Polyfluoroalkyl Substances (Pfas) in Belgian Wastewater Using Target, Suspect and Non-Target Screening Approaches

Non-Targeted Screening Approaches for Profiling of Volatile Organic Compounds Based on Gas Chromatography-Ion Mobility Spectroscopy (GC-IMS) and Machine Learning.

Due to its high sensitivity and resolving power, gas chromatography-ion mobility spectrometry (GC-IMS) is a powerful technique for the separation and sensitive detection of volatile organic compounds. It is a robust and easy-to-handle technique, which has recently gained attention for non-targeted screening (NTS) approaches. In this article, the general working principles of GC-IMS are presented. Next, the workflow for NTS using GC-IMS is described, including data acquisition, data processing and model building, model interpretation and complementary data analysis. A detailed overview of recent studies for NTS using GC-IMS is included, including several examples which have demonstrated GC-IMS to be an effective technique for various classification and quantification tasks. Lastly, a comparison of targeted and non-targeted strategies using GC-IMS are provided, highlighting the potential of GC-IMS in combination with NTS.

Removal of drug losartan in environmental aquatic matrices by heat-activated persulfate: Kinetics, transformation products and synergistic effects

In this study, the oxidative degradation of losartan (LOS), a widely administered medicine for high blood pressure by heat-activated persulfate was investigated. Increased temperature and persulfate concentration, as well as acidic conditions enhance the degradation efficiency of LOS, whose rate follows pseudo-first order kinetics. From the respective apparent rate constants in the range 40–60 °C, an apparent activation energy of 112.70 kJ/mol was computed. Radical scavenging tests demonstrated that both HO• and SO4•− contribute towards LOS degradation. LOS degradation was suppressed in real water matrices including bottled water (BW) and secondary wastewater effluent (WW), while other experiments indicated that the presence of bicarbonates and humic acid negatively affected its oxidation. Instead, the addition of chloride ions at 250 mg/L resulted in a positive effect on LOS removal. The combination of heat-activated PS with low-frequency ultrasound exhibited a synergistic effect, with the ratio S being 2.29 in BW and 1.52 in WW. Five transformation products of LOS were identified through HRMS suspect and non-target screening approaches, among which two are reported for the first time. Using the in-house risk assessment program, ToxTrAMs was revealed that most of the identified TPs present higher toxicity than LOS against Daphnia magna.

Micropollutants in four Brazilian water reservoirs

The concern about emerging contaminants such as pharmaceuticals is growing, mainly due to the increased global consumption of synthetic chemicals and the potential risk to environmental and human health. Although developing countries may be hotspots of pharmaceutical pollution, the knowledge about the occurrence of pharmaceuticals is still limited and patchy. Brazil holds one of the largest freshwater volumes globally, yet, little is known about the occurrence of pharmaceuticals in reservoirs although they make up key water sources. The aim of this study was, therefore, to investigate micropollutant occurrence, mainly pharmaceuticals, in four freshwater reservoirs distributed in Brazil. Water samples were collected in the Curuá-Una (CUN, Amazon region), Chapéu D’Uvas (CDU, Atlantic Forest region), Funil (FUN, Atlantic Forest region), and Simplício (SIM, Atlantic Forest region) reservoirs. The occurrence of 28 different micropollutants, including 26 pharmaceuticals, was investigated with target analysis on a UHPLC-Orbitrap-MS/MS, and a non-target screening approach was performed on all water samples to identify the presence of additional contaminants. The highest micropollutant concentrations were observed in FUN and SIM, which are the reservoirs with the largest population size in the catchment. Only caffeine was detected in CDU and CUN, which are reservoirs less influenced by urbanization. Metformin was the pharmaceutical with the highest concentrations, reaching 2 191 ng L−1 in FUN. The non-target screening identified 125 chemicals, of which most were pharmaceuticals. The numbers of compounds identified and which were above the LOQ were higher in FUN and SIM, in agreement with results from the target analysis. Metformin is the compound with the highest risk to affect FUN reservoir negatively, based on calculated risk quotients. Considering that the reservoirs are used for multiple purposes, including water supply, irrigation, and aquaculture, it is important to continue investigating micropollutant occurrence to guarantee environmental and human health.

Identification of Erythromycin and Clarithromycin Metabolites Formed in Chicken Liver Microsomes Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Nontargeted analysis can be used for the rapid screening and confirmatory analysis of veterinary drugs and their metabolites, which are important for the comprehensive safety evaluation of animal-derived foods. Here, a novel nontargeted screening approach based on liquid chromatography coupled with electrospray ionization–high-resolution mass spectrometry (LC/ESI–HR-MS) was developed to determine erythromycin, clarithromycin, and their metabolites in chicken liver microsomes. Erythromycin and clarithromycin were incubated in vitro in the presence of NADPH for 60 min to generate metabolites in chicken liver microsomes. After the incubation, the supernatant was extracted using ultrasonic shaking, orbital shaking, and centrifugation before analysis using LC/ESI-HR-MS in positive ion mode on an Agilent Eclipse Plus C18 column (100 mm × 2.1 mm; i.d. 3.5 µm) with 0.1 percent formic acid-water and acetonitrile as the mobile phases for gradient elution at 0.4 mL/min. The results show that erythromycin can produce N-desmethyl-erythromycin A in chicken liver microsomes, but clarithromycin cannot produce N-desmethyl-clarithromycin in chicken liver microsomes. The N-desmethyl-erythromycin A and N-desmethyl-clarithromycin were tentatively identified in chicken liver microsomes using the established quick analytic method, which will provide a theoretical foundation for future research on pharmacokinetics and drug elimination in poultry.

First insight on in vitro metabolism of three newly identified aryl organophosphate esters via a suspect coupled with nontarget screening approach

The ubiquity of organophosphate esters (OPEs) in the environment has triggered research into metabolic pathways of OPEs. Using liquid chromatography coupled with a hybrid quadrupole Orbitrap high-resolution mass spectrometer, a suspect and characteristic fragment ion-based nontarget screening strategy for the identification of unknown OPE metabolites was developed and evaluated. Then, this integrated approach was successfully used for investigation of three newly identified organophosphate esters (NOPEs), namely 2-biphenylol diphenyl phosphate (BPDPP), tris(2-biphenyl) phosphate (TBPHP), and naphthalen-2-yl diphenyl phosphate (NDPHP), in human liver microsomes (HLMs). The results demonstrated that BPDPP, TBPHP, and NDPHP were effectively metabolized by HLMs, with zero-order kinetics (R2 = 0.48−0.94) within the time frame of the assay. The suspected approach identified a considerable number of dearylated phosphate (DP), and hydroxylated metabolites for each of NOPEs after incubation with HLMs for 2 h. In addition, the nontarget approach further identified 9 novel metabolites including 2 epoxide intermediates and 7 oxidative ring-opening compounds, which were first reported in the Phase I metabolism of OPEs. Collectively, this study provided a novel suspect coupled with nontarget screening approach and was successfully used to screen metabolites of three NOPEs. For the first time, we observed direct evidence that oxidative ring-opening might serve as another primary metabolic pathway regarding the metabolism of aryl OPEs.

Identification of biotransformation products of organophosphate ester from various aquatic species by suspect and non-target screening approach

Organic pollutants that are introduced into the aquatic ecosystem can transform by various mechanisms. Biotransformation is an important process for predicting the remaining structures of pollutants in the ecosystem, and their toxicity. This study focused on triphenyl phosphate (TPHP), which is a commonly used organophosphate flame retardant and plasticizer. Since TPHP is particularly toxic to aquatic organisms, it is essential to understand its biotransformation in the aquatic environment. In the aquatic ecosystem, based on consideration of the producer-consumer-decomposer relationship, the biotransformation products of TPHP were identified, and their toxicity was predicted. Liquid chromatography-high resolution mass spectrometry was used for target, suspect, and non-target analysis. The obtained biotransformation products were estimated for toxicity based on the prediction model. As a result, 29 kinds of TPHP biotransformation products were identified in the aquatic ecosystem. Diphenyl phosphate was detected as a common biotransformation product through a hydrolysis reaction. In addition, products were identified by the biotransformation mechanisms of green algae, daphnid, fish, and microorganism. Most of the biotransformation products were observed to be less toxic than the parent compound due to detoxification except some products (hydroquinone, beta-lyase products, palmitoyl/stearyl conjugated products). Since various species exist in a close relationship with each other in an ecosystem, an integrated approach for not only single species but also various connected species is essential.

Metabolite fingerprinting of oil palm (Elaeis guineensis Jacq.) root for the identification of altered metabolic pathways associated with basal stem rot (BSR) disease

Basal stem rot (BSR) disease caused by Ganoderma boninense has remained as one of the major obstacles in oil palm (Elaeis guineensis Jacq.) plantation. BSR is a destructive disease, which contributes to significant yield losses and impacts to the oil palm industry. However, to date, there is still no effective control of the disease. Metabolite fingerprinting is an established non-targeted screening approach to classify samples based on metabolite patterns that change in response to G. boninense infection in oil palm. This study aimed to compare the metabolic profiles of natural Ganoderma-infected and healthy control oil palm root samples using liquid chromatography-quadrupole/time-of-flight-mass spectrometry (LC-Q/TOF-MS) combined with multivariate data analysis (MVDA) and to identify potential metabolic pathway(s) involved in response to BSR. MVDA revealed differential metabolites from oil palm root associated with natural Ganoderma-infected vs. healthy oil palms. A systematic metabolic pathway analysis of differential metabolites discovered the significant involvement of amino acid metabolism, carbohydrate metabolism and biosynthesis of other secondary metabolites in response to BSR disease. Unravelling the metabolic mechanisms involved during pathogen attack provides new knowledge and fill gaps in the information related to the oil palm-Ganoderma pathosystem.

Development of Antioxidant Protein Extracts from Gilthead Sea Bream (Sparus aurata) Side Streams Assisted by Pressurized Liquid Extraction (PLE).

The pressurized liquid extraction (PLE) technique was used, for the first time, to obtain protein extracts with antioxidant activity from side streams (muscle, heads, viscera, skin, and tailfins) of gilthead sea bream (Sparus aurata) in order to give added value to these underutilized matrices. Extraction conditions previously optimized for sea bass (Dicentrarchus labrax) side streams were applied. Protein recovery percentages were 22% (muscle), 33% (heads), 78% (viscera), 24% (skin), and 26% (tailfins), which represented an increase of 1.2–4.5-fold compared to control samples (extraction by stirring). The SDS-PAGE profiles revealed that PLE-assisted extraction influenced protein molecular weight distribution of the obtained extracts. PLE conditions also allowed increasing the antioxidant capacity measured by both Trolox equivalent antioxidant capacity (TEAC; 1.3–2.4 fold) and oxygen radical absorbance capacity (ORAC; 1.9–6.4) assays for all fish extracts. Inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-qTOF-MS) were used to investigate the presence of toxic metals and mycotoxins in sea bream side streams. The levels of As, Hg, Cd, and Pb were below those established by authorities for fish muscle for human consumption (except for Cd in viscera samples). Through a nontargeted screening approach, no mycotoxins or related metabolites were detected for all sea bream side streams. This study contributes to the research on the valorization of fish processing side streams using environmentally friendly technology.

Recent advancements in LC-MS based analysis of biotoxins: Present and future challenges.

There has been a rising concern regarding the harmful impact of biotoxins, source of origin, and the determination of the specific type of toxin. With numerous reports on their extensive spread, biotoxins pose a critical challenge to figure out their parent groups, metabolites, and concentration. In that aspect, liquid chromatography-mass spectrometry (LC-MS) based analysis paves the way for its accurate identification and quantification. The biotoxins are ideally categorized as phytotoxins, mycotoxins, shellfish-toxins, ciguatoxins, cyanotoxins, and bacterial toxins such as tetrodotoxins. Considering the diverse nature of biotoxins, both low-resolution mass spectrometry (LRMS) and high-resolution mass spectrometry (HRMS) methods have been implemented for their detection. The sample preparation strategy for complex matrix usually includes "QuEChERS" extraction or solid-phase extraction coupled with homogenization and centrifugation. For targeted analysis of biotoxins, the LRMS consisting of a tandem mass spectrometer operating in multiple reaction monitoring mode has been widely implemented. With the help of the reference standard, most of the toxins were accurately quantified. At the same time, the suspect screening and nontarget screening approach are facilitated by the HRMS platforms during the absence of reference standards. Significant progress has also been made in sampling device employment, utilizing novel sample preparation strategies, synthesizing toxin standards, employing hybrid MS platforms, and the associated data interpretation. This critical review attempts to elucidate the progress in LC-MS based analysis in the determination of biotoxins while pointing out major challenges and suggestions for future development.