Suspect Screening Workflow Research Articles

Identification of candidate exposure biomarkers for acetyl tributyl citrate and acetyl triethyl citrate using suspect screening in human liver microsomes

Acetyl tributyl citrate (ATBC) and acetyl triethyl citrate (ATEC) are increasingly used as alternatives to phthalates in various products, including food packaging, medical devices, and personal care items, raising concerns about their potential health impacts. This study aimed to investigate the in vitro human metabolism of ATBC and ATEC and identify potential exposure biomarkers applicable in human biomonitoring. Pooled human liver microsomes were utilized to conduct in vitro metabolism assays of deuterium labeled ATBC (ATBC-d3) and ATEC, and ultra performance liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry (UPLC-qToF/MS) was employed for analysis. Suspect screening workflow and confidence level assignment were applied for metabolite identification. Time-course analysis revealed rapid metabolism of both compounds, with estimated apparent half-lives of approximately 5 min for ATBC-d3 and less than 15 min for ATEC. Eleven metabolites were identified for ATBC-d3 and six for ATEC. The predominant chemical reactions observed were carboxylic ester hydrolysis, deacetylation, and hydroxylation. Based on their abundance and specificity, MB1 (hydroxylated) and MB11 (hydrolyzed and hydroxylated) were proposed as candidate exposure biomarkers for ATBC, and ME1 (hydrolyzed and deacetylated) for ATEC. The identified metabolites and proposed sequences of kinetic process enhance our understanding of the fate of these compounds in the human body, potentially informing future toxicological assessments and guiding the development of more comprehensive human biomonitoring strategies.

Wristband Personal Passive Samplers and Suspect Screening Methods Highlight Gender Disparities in Chemical Exposures.

Wristband personal samplers enable human exposure assessments for a diverse range of chemical contaminants and exposure settings with a previously unattainable scale and cost-effectiveness. Paired with nontargeted analyses, wristbands can provide important exposure monitoring data to expand our understanding of the environmental exposome. Here, a custom scripted suspect screening workflow was developed in the R programming language for feature selection and chemical annotations using gas chromatography-high-resolution mass spectrometry data acquired from the analysis of wristband samples collected from five different cohorts. The workflow includes blank subtraction, internal standard normalization, prediction of chemical uses in products, and feature annotation using multiple library search metrics and metadata from PubChem, among other functionalities. The workflow was developed and validated against 104 analytes identified by targeted analytical results in previously published reports of wristbands. A true positive rate of 62 and 48% in a quality control matrix and wristband samples, respectively, was observed for our optimum set of parameters. Feature analysis identified 458 features that were significantly higher on female-worn wristbands and only 21 features that were significantly higher on male-worn wristbands across all cohorts. Tentative identifications suggest that personal care products are a primary driver of the differences observed.

Prioritization, Identification, and Quantification of Emerging Contaminants in Recycled Textiles Using Non-Targeted and Suspect Screening Workflows by LC-ESI-HRMS.

Recycled textiles are becoming widely available to consumers as manufacturers adopt circular economy principles to reduce the negative impact of garment production. Still, the quality of the source material directly impacts the final product, where the presence of harmful chemicals is of utmost concern. Here, we develop a risk-based suspect and non-targeted screening workflow for the detection, identification, and prioritization of the chemicals present in consumer-based recycled textile products after manufacture and transport. We apply the workflow to characterize 13 recycled textile products from major retail outlets in Sweden. Samples were extracted and analyzed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS). In positive and negative ionization mode, 20,119 LC-HRMS features were detected and screened against persistent, mobile, and toxic (PMT) as well as other textile-related chemicals. Six substances were matched with PMT substances that are regulated in the European Union (EU) with a Level 2/3 confidence. Forty-three substances were confidently matched with textile-related chemicals reported for use in Sweden. For estimating the relative priority score, aquatic toxicity and concentrations were predicted for 7416 features with tandem mass spectra (MS2) and used to rank the non-targeted features. The top 10 substances were evaluated due to elevated environmental risk linked to the recycling process and potential release at end-of-life.

Insights into the early-life chemical exposome of Nigerian infants and potential correlations with the developing gut microbiome

Early-life exposure to natural and synthetic chemicals can impact acute and chronic health conditions. Here, a suspect screening workflow anchored on high-resolution mass spectrometry was applied to elucidate xenobiotics in breast milk and matching stool samples collected from Nigerian mother-infant pairs (n = 11) at three time points. Potential correlations between xenobiotic exposure and the developing gut microbiome, as determined by 16S rRNA gene amplicon sequencing, were subsequently explored. Overall, 12,192 and 16,461 features were acquired in the breast milk and stool samples, respectively. Following quality control and suspect screening, 562 and 864 features remained, respectively, with 149 of these features present in both matrices. Taking advantage of 242 authentic reference standards measured for confirmatory purposes of food bio-actives and toxicants, 34 features in breast milk and 68 features in stool were identified and semi-quantified. Moreover, 51 and 78 features were annotated with spectral library matching, as well as 416 and 652 by in silico fragmentation tools in breast milk and stool, respectively. The analytical workflow proved its versatility to simultaneously determine a diverse panel of chemical classes including mycotoxins, endocrine-disrupting chemicals (EDCs), antibiotics, plasticizers, perfluorinated alkylated substances (PFAS), and pesticides, although it was originally optimized for polyphenols. Spearman rank correlation of the identified features revealed significant correlations between chemicals of the same classification such as polyphenols. One-way ANOVA and differential abundance analysis of the data obtained from stool samples revealed that molecules of plant-based origin elevated as complementary foods were introduced to the infants’ diets. Annotated compounds in the stool, such as tricetin, positively correlated with the genus Blautia. Moreover, vulgaxanthin negatively correlated with Escherichia-Shigella. Despite the limited sample size, this exploratory study provides high-quality exposure data of matched biospecimens obtained from mother-infant pairs in sub-Saharan Africa and shows potential correlations between the chemical exposome and the gut microbiome.

High resolution mass spectrometry targeted analysis and suspect screening of pesticide residues in fruit samples and assessment of dietary exposure

Fruits consistently hold a prominent position in healthy dietary habits. Pesticides are used to manage plant diseases, achieve sustainable production, and maintain high food standards. This study utilized a comprehensive analytical technique that involved both targeted analysis and suspect screening. Analysis was conducted using Ultra-high-performance liquid chromatography coupled with hybrid Linear Trap Quadrupole (LTQ)/Orbitrap High Resolution Mass Spectrometry (HRMS) to examine pesticide levels in fruits. The matrices chosen comprised fruit commodities that are commonly consumed in Greece, including table grapes, apples, pears, citrus fruits, and strawberries. The QuEChERS approach was effectively validated for 30 specific pesticides. According to the method acceptance criteria established by SANTE, the QuEChERS method have shown exceptional efficiency in extracting the chosen pesticides, with recovery rates ranging from 70% to 120% in three concentration levels (10, 50, 100 μg kg−1). It also exhibited outstanding linearity, with an R2 more than 0.99. The method exhibited exceptional precision, with relative standard deviations (RSDs) below 20%. Additionally, the combined measurement uncertainty (MU%) was found to be acceptable, remaining below 50%The quantification limits were below 10 μg kg−1 for the majority of the analytes, satisfying the Maximum Residue Levels (MRLs) established by the European Commission. Following targeted analysis, a dietary risk assessment was performed, revealing that both acute and chronic hazard quotients (aHQ and cHQ), along with chronic hazard index (cHI) were below 1, which indicated that the studied commodities are safe for human consumption. In addition, a suspect screening workflow was developed based on an in-house database comprising 355 pesticides commonly applied to the relevant commodities and related transformation products (TPs). Overall, through suspect screening, twenty-two additional pesticides and TPs not included in the target list were identified. Hence, this approach is anticipated to function as proactive alert system guaranteeing the long-term viability of agricultural production.

Unravelling psychoactive substances and their metabolites and transformation products: High-Resolution Mass Spectrometry approaches for comprehensive target and suspect screening in wastewater

Untangling the consumption rates of psychiatric drugs and their metabolites/ transformation products-(TPs) through wastewater gains attention lately. However, the potential environmental impact caused by their release remains ambiguous. As it follows, the monitoring of this class of pharmaceuticals as well as the evaluation of their potential toxicity is a matter of high concern. In the light of the above, here, wastewater samples, were collected in a 1-year and a half sampling campaign (2020−2021) and were further subjected to solid phase extraction. A Q Exactive Focus Orbitrap mass analyzer was employed for the analysis of the samples. For the data curation, except of the monitoring of targets, a comprehensive suspect screening workflow was developed and slightly optimized based on a lab made HRMS database for the investigation of legally or illegally prescribed psychiatric drugs and their relevant metabolites/TPs in influents and effluents. Carbamazepine and amisulpride were quantified at the highest mean concentrations 243 and 225 ng/L respectively, in influents. In effluents, the highest mean concentrations were calculated for carbamazepine (180 ng/L) and venlafaxine (117 ng/L). The implementation of suspect screening approach enhanced the comprehensiveness of analysis by detecting 29 compounds not included in the target list. O-Desmethylvenlafaxine was the predominant metabolite in influents presenting a mean concentration equal to 87 ng/L while the same pattern was also noticed in effluents where the mean concentration was up to 91 ng/L. From the group of suspect compounds for which no analytical standards were available, the predominant compounds with detection frequency 100 % were norephedrine and codeine in influents while in effluents, oxazepam was detected in 81 % of the analyzed samples. Finally, in silico and mathematical tools were employed for the assessment of the risk posed to environmental systems. Most of the detected compounds present high risk in all trophic levels.

Comprehensive polyphenolic profiling of nine distinct plants and edible mushrooms by targeted and untargeted LC-(HR)MS(/MS)

Polyphenols are a broad molecular family whose occurence patterns in an organism are often only partially known. They are secondary metabolites exhibiting antioxidant properties and are involved in a wide range of biological actions. Moreover, polyphenol oxidases, metalloenzymes present in various plants and fungi, play a role in metabolic pathways by catalyzing the hydroxylation and oxidation of polyphenols. Functional studies on these enzymes are greatly hampered by the lack of information on the polyphenols present in a specific organisms, meaning the physiological substrate is unknown for most polyphenol oxidases. Therefore, seven commercially available fungal and two plant species were examined in detail for the presence of polyphenols using an LC-HRMS-based suspect screening workflow. A total of 401 features were annotated, composed of 221 flavonoids and 180 non-flavonoids. As 64% of the flavonoids were conjugated with a glycone, (semi-)quantification was applied when a reference standard of the aglycone was available. Isomers of hesperetin-O-glucoside were ubiquitous in all investigated samples. The samples were then additionally investigated by a quantitative, targeted LC-MS/MS assay that covers 90 polyphenols from 13 distinct polyphenol classes. The targeted assay showed phenolic acids as prevalent analytes. Overall, this pilot study provides new insights into the complex polyphenol profiles of nine mushroom and plant species.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography – high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

Development and evaluation of a comprehensive workflow for suspect screening of exposome-related xenobiotics and phase II metabolites in diverse human biofluids

Suspect and non-target screening (SNTS) methods are being promoted in order to decode the human exposome since a wide chemical space can be analysed in a diversity of human biofluids. However, SNTS approaches in the exposomics field are infra-studied in comparison to environmental or food monitoring studies. In this work, a comprehensive suspect screening workflow was developed to annotate exposome-related xenobiotics and phase II metabolites in diverse human biofluids. Precisely, human urine, breast milk, saliva and ovarian follicular fluid were employed as samples and analysed by means of ultra-high performance liquid chromatography coupled with high resolution tandem mass spectrometry (UHPLC-HRMS/MS). To automate the workflow, the “peak rating” parameter implemented in Compound Discoverer 3.3.2 was optimized to avoid time-consuming manual revision of chromatographic peaks. In addition, the presence of endogenous molecules that might interfere with the annotation of xenobiotics was carefully studied as the employment of inclusion and exclusion suspect lists. To evaluate the workflow, limits of identification (LOIs) and type I and II errors (i.e., false positives and negatives, respectively) were calculated in both standard solutions and spiked biofluids using 161 xenobiotics and 22 metabolites. For 80.3 % of the suspects, LOIs below 15 ng/mL were achieved. In terms of type I errors, only two cases were identified in standards and spiked samples. Regarding type II errors, the 7.7 % errors accounted in standards increased to 17.4 % in real samples. Lastly, the use of an inclusion list for endogens was favoured since it avoided 18.7 % of potential type I errors, while the exclusion list caused 7.2 % of type II errors despite making the annotation workflow less time-consuming.

Establishment of an Integrated Nontarget and Suspect Screening Workflow for Organophosphate Diesters (Di-OPEs) and Identification of Seven Previously Unknown Di-OPEs in Food Contact Plastics.

In this study, an innovative, integrated nontarget and suspect screening workflow was developed for identifying organophosphate diesters (di-OPEs) using high-resolution mass spectrometry. The workflow featured the utilization of 0.02% acetic acid as a mobile-phase additive, differentiated screening methods for alkyl and aryl di-OPEs, and a combination of electrospray negative ionization and positive ionization. Using this workflow, 18 di-OPEs were identified in the extracts of 75 food contact plastic (FCP) samples sourced from South China. Among these, six alkyl and one aryl di-OPEs were previously unknown (one unequivocal identification and six probable structures based on diagnostic evidence). (Semi)quantification revealed that bis(2,4-di-tert-butylphenyl) phosphate was the major di-OPE in FCPs, with a median concentration of 1079 ng/g (range: 23.4-158,414 ng/g). The migration efficiencies of di-OPEs from an FCP sample to four kinds of food simulants were between 2.58 and 54.3%. This study offered a useful workflow for the comprehensive profiling of di-OPEs in FCPs.

Nontargeted Screening of Contaminants of Emerging Concern in the Glen Valley Wastewater Treatment Plant, Botswana.

There is growing concern about the prevalence and impact of contaminants of emerging concern (CECs). The environmental monitoring of CECs has, however, been limited in low- and middle-income countries due to the lack of advanced analytical instrumentation locally. In the present study we employed a nontargeted and suspect screening workflow via liquid chromatography coupled with high-resolution mass spectrometry (HRMS) to identify known and unknown pollutants in the Glen Valley wastewater treatment plant, Botswana, complemented by analysis of groundwater samples. The present study represents the first HRMS analysis of CECs in water samples obtained in Botswana. Suspect screening of 5942 compounds qualitatively identified 28 compounds, including 26 pharmaceuticals and two illicit drugs (2-ethylmethcathinone and 11-nor-9-carboxy-Δ9-tetrahydrocannabinol). Nontargeted analysis tentatively identified the presence of 34 more compounds including (5ξ)-12,13-dihydroxypodocarpa-8,11,13-trien-7-one, 12-aminododecanoic acid, atenolol acid, brilliant blue, cyclo leucylprolyl, decanophenone, DL-carnitine, N,N'-dicyclohexylurea, N4-acetylsulfamethoxazole, NP-003672, and 24 polyethylene glycol polymers. The highest number of detections were in influent wastewater (26 CECs) followed by effluent wastewater (10 CECs) and, lastly, groundwater (4 CECs). Seventeen CECs detected in the influent water were not detected in the effluent waters, suggesting reduced emissions due to wastewater treatment. Two antiretroviral compounds (abacavir and tenofovir) were detected in the influent and effluent sources. This suggests that wastewater treatment plants are a major pathway of chemical pollution to the environment in Botswana and will help inform prioritization efforts for monitoring and remediation that is protective of these key ecosystems. Environ Toxicol Chem 2024;43:52-61. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Assessment of exposure to pesticides: residues in 24 h duplicate diets versus their metabolites in 24 h urine using suspect screening and target analysis

Human biomonitoring can add value to chemical risk assessment by reducing the assumptions regarding consumption rates, residue occurrence, and processing effects and by integrating exposures from different sources (diet, household use, environmental). However, the relationship between exposure and concentration in human matrices is unknown for most pesticides. Therefore, we conducted a pilot study to gain more insight into the qualitative and quantitative relationship between dietary intake of pesticides (external exposure) and urinary excretion (reflecting internal exposure). In this cross-sectional observational study, 35 healthy consumers aged 18–65 years from the region of Wageningen, Netherlands, collected an exact duplicate portion of their diets during 24 h. On the same day, they also collected all their urine. The duplicate diets were analyzed using target screening by GC- and LC-HRMS; each duplicate diet contained at least five, up to 21, pesticide residues. The 24 h urine samples were analyzed using LC-HRMS in a suspect screening workflow. Metabolites were tentatively detected in all 24 h urine samples, ranging from six metabolites corresponding to four pesticides up to 40 metabolites originating from 16 pesticides in a single urine sample. In total, 65 metabolites originating from 28 pesticides were tentatively detected. After prioritization and additional confirmation experiments, 28 metabolites originating from 10 pesticides were identified with confidence level 1 or 2b. Next, quantitative analysis was performed for a selection of pesticides in duplicate diets and their metabolites in 24 h urine to assess quantitative relationships. In the quantitative comparisons between duplicate diet and 24 h urine, it was found that some metabolites were already present in the duplicate diet, which may give an overestimation of exposure to the parent pesticide based on measurement of the metabolites in urine. Additionally, the quantitative comparisons suggest a background exposure through other exposure routes. We conclude that suspect screening of 24 h urine samples can disclose exposure to mixtures of pesticide on the same day in the general population. However, more research is needed to obtain quantitative relationships between dietary intake and exposure.Graphical

A method for the sensitive targeted screening of synthetic cannabinoids and opioids in whole blood by LC-QTOF-MS with simultaneous suspect screening using HighResNPS.com.

A sensitive method for the qualitative screening of synthetic cannabinoids and opioids in whole blood was developed and validated using alkaline liquid-liquid extraction (LLE) and liquid chromatography - time-of-flight mass spectrometry (LC-QTOF-MS). Estimated limits of detection for validated compounds ranged from 0.03-0.29 µg/L (median, 0.04 µg/L) for the 27 opioids and from 0.04-0.5 µg/L (median, 0.07 µg/L) for the 23 synthetic cannabinoids. Data processing occurred in two stages; first a targeted screen was performed using an in-house database containing retention times, accurate masses and MS/MS spectra for 79 cannabinoids and 53 opioids. Suspect screening was then performed using a database downloaded from the crowd sourced NPS data website HighResNPS.com which contains mass, consensus MS/MS data and laboratory-specific predicted retention times for a far greater number of compounds. The method was applied to 61 forensic cases where synthetic cannabinoid or opioid screening was requested by the client or their use was suspected due to case information. CUMYL-PEGACLONE was detected in two cases and etodesnitazine, 5F-MDMB-PICA, 4-cyano-CUMYL-BUTINACA and carfentanil were detected in one case each. These compounds were within the targeted scope of the method but were also detected through the suspect screening workflow. The method forms a solid base for expansion as more compounds emerge onto the illicit drug market.

Comprehensive HRMS Chemical Characterization of Pomegranate-Based Antioxidant Drinks via a Newly Developed Suspect and Target Screening Workflow.

Antioxidants play a significant role in human health, protecting against a variety of diseases. Therefore, the development of products with antioxidant activity is becoming increasingly prominent in the human lifestyle. New antioxidant drinks containing different percentages of pomegranate, blackberries, red grapes, and aronia have been designed, developed, and manufactured by a local industry. The comprehensive characterization of the drinks' constituents has been deemed necessary to evaluate their bioactivity. Thus, LC-qTOFMS has been selected, due to its sensitivity and structure identification capability. Both data-dependent and -independent acquisition modes have been utilized. The data have been treated according to a novel, newly designed workflow based on MS-DIAL and MZmine for suspect, as well as target screening. The classical MS-DIAL workflow has been modified to perform suspect and target screening in an automatic way. Furthermore, a novel methodology based on a compiled bioactivity-driven suspect list was developed and expanded with combinatorial enumeration to include metabolism products of the highlighted metabolites. Compounds belonging to ontologies with possible antioxidant capacity have been identified, such as flavonoids, amino acids, and fatty acids, which could be beneficial to human health, revealing the importance of the produced drinks as well as the efficacy of the new in-house developed workflow.

Does climbazole instigate a threat in the environment as persistent, mobile and toxic compound? Unveiling the occurrence and potential ecological risks of its phototransformation products in the water cycle

Persistent, mobile, and toxic chemicals (PMT), such as the antimycotic climbazole-(CBZ), proliferate in water cycle and imperil drinking water quality, sparking off research about their environmental fate. Unlike the parent compound, its transformation products-(TPs) are scarcely investigated, much less as PMTs. To this end, phototransformation of CBZ was investigated. A novel suspect-screening workflow was developed and optimized by cross-comparing the results of the identified photo-TPs against literature data to create an enhanced HRMS-database for environmental investigations of CBZ/TPs in the water cycle. In total, 24 TPs were identified, 14 of which are reported for the first time. Isomerism, dechlorination, hydroxylation, and cleavage of the ether or C-N bond are suggested as the main transformation routes. A screening of CBZ/TPs was conducted in wastewater, leachates, surface, and groundwater, revealing a maximum concentration of 464.8 ng/L in groundwater. In silico and in vitro methods were used for toxicity assessment, indicating toxicity for CBZ and some TPs. Seemingly, CBZ is rightly considered as PMT, and a higher potential to occur in surface or groundwater than non-PM chemicals appears. Likewise, the occurrence of TPs due to PMT properties or emission patterns was evaluated.

InSpectra – A platform for identifying emerging chemical threats

Non-target analysis (NTA) employing high-resolution mass spectrometry (HRMS) coupled with liquid chromatography is increasingly being used to identify chemicals of biological relevance. HRMS datasets are large and complex making the identification of potentially relevant chemicals extremely challenging. As they are recorded in vendor-specific formats, interpreting them is often reliant on vendor-specific software that may not accommodate advancements in data processing. Here we present InSpectra, a vendor independent automated platform for the systematic detection of newly identified emerging chemical threats. InSpectra is web-based, open-source/access and modular providing highly flexible and extensible NTA and suspect screening workflows. As a cloud-based platform, InSpectra exploits parallel computing and big data archiving capabilities with a focus for sharing and community curation of HRMS data. InSpectra offers a reproducible and transparent approach for the identification, tracking and prioritisation of emerging chemical threats.

Advanced throughput analytical strategies for the comprehensive HRMS screening of organic micropollutants in eggs of different bird species

Raptors are ideal indicators for biomonitoring studies using wildlife in order to assess the environmental pollution in the terrestrial ecosystem, since they are placed in the highest trophic position in the food webs and their life expectancy is relatively long. In this study, 26 eggs of 4 bird species (Peregrine falcon, Eurasian curlew, Little owl and Eagle owl) collected in Germany, were investigated for the presence of persistent organic pollutants (POPs) and thousands of contaminants of emerging concern (CECs). Generic sample preparation protocols were followed for the extraction of the analytes and the purification of the extracts, and the samples were analyzed both by liquid (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS), for capturing a wide range of organic micropollutants with different physicochemical properties. State-of-the-art screening methodologies were applied in the acquired HRMS data, including wide-scope target analysis of 2448 known pollutants and suspect screening of over 65,000 environmentally relevant compounds. Overall, 58 pollutants from different chemical classes, such as plant protection products, per- and polyfluoroalkyl substances and medicinal products, as well as their transformation products, were determined through target analysis. Most of the detected compounds were lipophilic (logP>2), although the presence of (semi)polar contaminants should not be overlooked, underlying the need for holistic analytical approaches in environmental monitoring studies. p,p’-DDE, PCB 153 and PCB138, PFOS and methylparaben were the most frequently detected compounds. 50 additional substances were identified and semi-quantified through suspect screening workflows, including mainly compounds of industrial use with high production volume.

A method for the sensitive targeted screening of synthetic cannabinoids and opioids in whole blood by LC-QTOF with simultaneous suspect screening using HighResNPS.com

To develop and validate a qualitative screening method capable of detecting the low concentrations found in cases of synthetic cannabinoid and opioid consumption using a targeted scope, while simultaneously performing efficient suspect screening against a much larger database. Screening for synthetic cannabinoids and opioids in blood samples remains challenging due to the large number of substances available and the constantly changing market, as well as the low limits of detection required for some compounds. Many laboratories employ LC-QQQ when screening for these compound classes due to the ubiquity and sensitivity of those instruments but LC-HRMS instruments are similarly able to detect a large number of pre-defined analytes with high sensitivity and specificity, as well as acquiring full scan data which enables both suspect screening and retrospective screening if required. Analytes were extracted from 0.5 mL of whole blood using alkaline liquid-liquid extraction and data were acquired using data dependent acquisition on an Agilent 6545 QTOF. 28 opioids and 23 cannabinoids were validated for limit of detection, recovery, matrix effects, selectivity, extract stability and carryover. Data processing occurred in two stages; first a targeted screen was performed using an in-house database containing retention times (RT), accurate masses and MS/MS spectra for the validated compounds plus an additional 16 opioids and 34 cannabinoids (101 total compounds). Suspect screening was then performed using a database downloaded from the crowdsourced NPS data website HighResNPS.com which contains mass, consensus MS/MS spectra and laboratory specific predicted retention times for over 1400 compounds. The suspect screening workflow was assessed by determining the accuracy of the retention time prediction and MS/MS library matching against consensus spectra. For this workflow, qualifying parameters for initial presumptive identification included mass accuracy of ±4mDa, MS/MS match score greater than 20 and retention time difference to predicted RT of ±2 min. In practice, results of this suspect screen would be verified following purchase of authentic reference material for comparison. The method was applied to 66 forensic cases where synthetic cannabinoid or opioid screening was requested by the client or their use was suspected due to case information. Limits of detection ranged from 0.03–0.27 μg/L (median, 0.04 μg/L) for the 28 validated opioids and 0.04–0.5 (median, 0.07 μg/L) for the 23 synthetic cannabinoids. Recoveries were greater than 70% for all compounds except MDMB-CHMCZCA (63%) and desomorphine (42%). Synthetic cannabinoids or opioids were detected in seven of the 66 forensic cases. CUMYL-PEGACLONE and etodesnitazine were each detected in two cases and 5F-MDMB-PICA, 4-Cyano-CUMYL-BUTINACA and carfentanil were detected in one case each. These compounds were within the targeted scope of the method but were also detected through the suspect screening workflow. The developed method has similar limits of detections to other published methods using LC-QQQ instruments but has several additional advantages. The suspect screening workflow enables efficient, automated screening of data against the HighResNPS database in the absence of reference materials. The suspect screening parameters adopted were verified through the successful detection of compounds in spiked validation samples, as well as compounds known to be present in authentic samples. The data acquired is also suitable for retrospective screening as new compounds emerge. The dual approach of simultaneous targeted and suspect screening allows laboratories certainty in their coverage of a defined scope, as well as a “best attempt” to cover a much large number of compounds. This provides a good compromise, especially for laboratories where the cost of purchasing hundreds of NPS reference standards is prohibitive. The advantages of testing for these compound classes by HRMS over QQQ mean this instrumentation should be selected preferentially wherever possible when developing new methods.

Identification and semi-quantification of metabolites of new plasticizers in urine collected from flemish adults and children

A suspect screening workflow combined with a semi-quantification method was applied for the investigation of metabolites of the plasticizers di-propylene glycol dibenzoate (DiPGDB) and tri-n-butyl trimellitate (TBTM) in human urine collected from adults and children during winter (W) and summer (S) seasons. Liquid chromatography - quadrupole time of flight mass spectrometry (LC-QTOF-MS) was applied for the analyses. Two direct and one indirect metabolites of DiPGDB were identified: 3-(3-hydroxypropoxy) propyl benzoate (DiPGDB-M194), 3,4,5-trihydroxy-6-[3-(3-hydroxypropoxy) propoxy] oxane-2-carboxylic acid (DiPGDB-M310), hippuric acid (DiPGDB-M179) and one metabolite of TBTM: bis(butoxycarbonyl) benzoyloxy]-3,4,5-trihydroxyoxane-2-carboxylic acid (TBTM-M498). The identified metabolites were reported with levels of confidence (LoC) 2 and 3 and their concentrations were assessed using a semi-quantification approach. The respective concentration ranges for W and S samples were 0.20–42 ng/mL and 0.07–29 ng/mL for DiPGDB-M194, 2.5–1420 ng/mL and 5.0–2320 ng/mL for DiPGDB-M310, 230–10840 ng/mL and 320–8420 ng/mL for DiPGDB-M179, and 0.40–30 ng/mL and 0.65–30 ng/mL for TBTM-M498. The detection frequency order in urine samples was DiPGDB-M310 = DiPGDB-M179 (100%) >TBTM-M498 (44%) > DiPGDB-M194 (28%) for W and DiPGDB-M179 (99%)> DiPGDB-M310 (98%) > TBTM-M498 (57%) > DiPGDB-M194 (30%) for S. The identified metabolites DiPGDB-M310, DiPGDB-M194 and TBTM-M498 are potential biomarkers for the evaluation of human exposure to DiPGDB and TBTM. DiPGDB-M179 cannot be used for the same purpose due to its formation from compounds with multi-source origin. The application of the semi-quantification method could be useful for further studies where analytical standards are not available.

Thorough Investigation of the Phenolic Profile of Reputable Greek Honey Varieties: Varietal Discrimination and Floral Markers Identification Using Liquid Chromatography-High-Resolution Mass Spectrometry.

Honey is a highly consumed commodity due to its potential health benefits upon certain consumption, resulting in a high market price. This fact indicates the need to protect honey from fraudulent acts by delivering comprehensive analytical methodologies. In this study, targeted, suspect and non-targeted metabolomic workflows were applied to identify botanical origin markers of Greek honey. Blossom honey samples (n = 62) and the unifloral fir (n = 10), oak (n = 24), pine (n = 39) and thyme (n = 34) honeys were analyzed using an ultra-high-performance liquid chromatography hybrid quadrupole time-of-flight mass spectrometry (UHPLC-q-TOF-MS) system. Several potential authenticity markers were revealed from the application of different metabolomic workflows. In detail, based on quantitative targeted analysis, three blossom honey markers were found, namely, galangin, pinocembrin and chrysin, while gallic acid concentration was found to be significantly higher in oak honey. Using suspect screening workflow, 12 additional bioactive compounds were identified and semi-quantified, achieving comprehensive metabolomic honey characterization. Lastly, by combining non-targeted screening with advanced chemometrics, it was possible to discriminate thyme from blossom honey and develop binary discriminatory models with high predictive power. In conclusion, a holistic approach to assessing the botanical origin of Greek honey is presented, highlighting the complementarity of the three applied metabolomic approaches.