New PFASs Identified in AFFF Impacted Groundwater by Passive Sampling and Nontarget Analysis.

Monitoring contamination from per- and polyfluoroalkyl substances (PFASs) in water systems impacted by aqueous film-forming foams (AFFFs) typically addresses a few known PFAS groups. Given the diversity of PFASs present in AFFFs, current analytical approaches do not comprehensively address the range of PFASs present in these systems. A suspect-screening and nontarget analysis (NTA) approach was developed and applied to identify novel PFASs in groundwater samples contaminated from historic AFFF use. A total of 88 PFASs were identified in both passive samplers and grab samples, and these were dominated by sulfonate derivatives and sulfonamide-derived precursors. Several ultrashort-chain (USC) PFASs (≤C3) were detected, 11 reported for the first time in Australian groundwater. Several transformation products were identified, including perfluoroalkane sulfonamides (FASAs) and perfluoroalkane sulfinates (PFASis). Two new PFASs were reported (((perfluorohexyl)sulfonyl)sulfamic acid; m/z 477.9068 and (E)-1,1,2,2,3,3,4,5,6,7,8,8,8-tridecafluorooct-6-ene-1-sulfonic acid; m/z 424.9482). This study highlights that several PFASs are overlooked using standard target analysis, and therefore, the potential risk from all PFASs present is likely to be underestimated.

Influence of extraction windows for data-independent acquisition on feature annotation during suspect screening.

Non-target analysis (NTA) using high-resolution mass spectrometry is becoming a useful approach to screen for suspect and unknown chemicals. For comprehensive analyses, data-independent acquisition (DIA), like Sequential Windowed Acquisition of all THeoretical Mass Spectra (SWATH-MS) on Sciex instruments, is necessary, usually followed by library matching for feature annotation. The choice of parameters, such as acquisition window number and size, may influence the comprehensiveness of the suspect features detected. The goal of this study was to assess how mass spectrometric DIA settings may influence the ability to obtain confident annotations and identifications of features in environmental (river water, passive sample extract (PSE)), wastewater (unpreserved and acidified) and biological (urine) sample matrices. Each matrix was analysed using 11 different MS methods, with 5-15 variable size acquisition windows. True positive (TP) annotation (i.e., matching experimental and library spectra) rates were constant for PSE (40%) and highest for urine (18%), wastewater (34% and 36%, unpreserved and acidified, respectively) and river water (8%) when using higher numbers of windows (15). The number of annotated features was highest for PSE (12%) and urine (8.5%) when using more acquisition windows (9 and 14, respectively). Less complex matrices (based on average total ion chromatogram intensities) like river water, unpreserved and acidified wastewater have higher annotation rates (7.5%, 8% and 13.2%, respectively) when using less acquisition windows (5-6), indicating matrix dependency of optimum settings. Library scores varied widely for correct (scores between 6 and 100) as well as incorrect annotations (scores between 2 and 100), making it hard to define specific ideal cut-off values. Results highlight the need for properly curated libraries and careful optimization of SWATH-MS and other DIA methods for each individual matrix, finding the best ratio of total annotations to true positive, (i.e., correct) annotations to achieve best NTA results.

Is Nontarget Analysis Ready for Regulatory Application? Influence of Peak-Picking Algorithms on Data Analysis.

The use of peak-picking algorithms is an essential step in all nontarget analysis (NTA) workflows. However, algorithm choice may influence reliability and reproducibility of results. Using a real-world data set, the aim of this study was to investigate how different peak-picking algorithms influence NTA results when exploring temporal and/or spatial trends. For this, drinking water catchment monitoring data, using passive samplers collected twice per year across Southeast Queensland, Australia (n = 18 sites) between 2014 and 2019, was investigated. Data were acquired using liquid chromatography coupled to high-resolution mass spectrometry. Peak picking was performed using five different programs/algorithms (SCIEX OS, MSDial, self-adjusting-feature-detection, two algorithms within MarkerView), keeping parameters identical whenever possible. The resulting feature lists revealed low overlap: 7.2% of features were picked by >3 algorithms, while 74% of features were only picked by a single algorithm. Trend evaluation of the data, using principal component analysis, showed significant variability between the approaches, with only one temporal and no spatial trend being identified by all algorithms. Manual evaluation of features of interest (p-value <0.01, log fold change >2) for one sampling site revealed high rates of incorrectly picked peaks (>70%) for three algorithms. Lower rates (<30%) were observed for the other algorithms, but with the caveat of not successfully picking all internal standards used as quality control. The choice is therefore currently between comprehensive and strict peak picking, either resulting in increased noise or missed peaks, respectively. Reproducibility of NTA results remains challenging when applied for regulatory frameworks.

Novel Per- and Polyfluoroalkyl Substances Discovered in Cattle Exposed to AFFF-Impacted Groundwater.

The leaching of per- and polyfluoroalkyl substances (PFASs) from Australian firefighting training grounds has resulted in extensive contamination of groundwater and nearby farmlands. Humans, farm animals, and wildlife in these areas may have been exposed to complex mixtures of PFASs from aqueous film-forming foams (AFFFs). This study aimed to identify PFAS classes in pooled whole blood (n = 4) and serum (n = 4) from cattle exposed to AFFF-impacted groundwater and potentially discover new PFASs in blood. Thirty PFASs were identified at various levels of confidence (levels 1a-5a), including three novel compounds: (i) perfluorohexanesulfonamido 2-hydroxypropanoic acid (FHxSA-HOPrA), (ii) methyl((perfluorohexyl)sulfonyl)sulfuramidous acid, and (iii) methyl((perfluorooctyl)sulfonyl)sulfuramidous acid, belonging to two different classes. Biotransformation intermediate, perfluorohexanesulfonamido propanoic acid (FHxSA-PrA), hitherto unreported in biological samples, was detected in both whole blood and serum. Furthermore, perfluoroalkyl sulfonamides, including perfluoropropane sulfonamide (FPrSA), perfluorobutane sulfonamide (FBSA), and perfluorohexane sulfonamide (FHxSA) were predominantly detected in whole blood, suggesting that these accumulate in the cell fraction of blood. The suspect screening revealed several fluoroalkyl chain-substituted PFAS. The results suggest that targeting only the major PFASs in the plasma or serum of AFFF-exposed mammals likely underestimates the toxicological risks associated with exposure. Future studies of AFFF-exposed populations should include whole-blood analysis with high-resolution mass spectrometry to understand the true extent of PFAS exposure.

Evaluation and identification of chemical migrants leached from baby food pouch packaging.

Multilayer plastic pouch (MLPP) has an estimated 9% annual increase of market growth. However, the migrants it can introduce to food has not yet been systematically studied. A total of 79 MLPPs for baby food were purchased from major retail outlets in Australia. The methodology for testing chemical migration followed the design of previous studies using four types of selected simulants according to the European Committee Regulation No. 10/2011 method. Four bisphenols and five phthalic acid diesters (PAEs) were detected, including the ones known for endocrine disrupting effect in human. Three intentionally added and 23 non-intentionally added substances (NIAS) were tentatively identified through a suspect screening procedure. Out of the 23 NIAS, neopentyl glycol - phthalic acid - 1,6-hexanediol - phthalic acid oligomer was identified for the first time with MLPP. A further two NIAS were detected for the first time in baby food related products. For 40% of the pouches where adipic acid - diethylene glycol was detected, the estimated exposure from consuming one pouch of food per day may exceed the threshold of toxicological concern established based on the Cramer classification.

Identification of novel unique manuka honey markers using high-resolution mass spectrometry-based metabolomics.

Manuka honey is a valuable commodity produced by bees foraging the flowers of Leptospermum scoparium, a bush native to New Zealand and Australia. Due to its high value and proven health benefits, authenticity fraud in the sale of this food is a significant risk, as recounted in the literature. Four compulsory natural products must be present at minimum concentrations to authenticate manuka honey (3-phenyllactic acid, 2'-methoxyacetophenone, 2-methoxybenzoic acid, and 4-hydroxyphenyllactic acid). However, spiking other kinds of honey with these compounds and/or the dilution of manuka honey with other varieties may result in fraud going undetected. In this work, liquid chromatography coupled with high-resolution mass spectrometry and a metabolomics-based strategy has allowed us to tentatively identify 19 natural products -putative manuka honey markers-, nine of which are reported for the first time. Chemometric models applied to these markers allowed the detection of both spiking and dilution fraud attempts of manuka honey, even at 75% manuka honey purity. Thus, the herein-reported methodology can be employed in the prevention and detection of manuka honey adulteration even at low levels, and the tentatively identified markers presented in this work proved valuable for manuka honey authentication procedures.

Cooking food in microwavable plastic containers: in situ formation of a new chemical substance and increased migration of polypropylene polymers.

Microwavable plastic food containers can be a source of toxic substances. Plastic materials such as polypropylene polymers are typically employed as safe materials in food packaging, but recent research demonstrates the migration of plastic substances or their by-products to food simulants, to foodstuff, and, more recently, to the human body through food consumption. However, a thorough evaluation of foodstuff in food contact materials under cooking conditions has not yet been undertaken. Here we show for the first time that plastic migrants present in food contact materials can react with natural food components resulting in a compound that combines a UV-photoinitiator (2-hydroxy-2-methyl-1-phenylpropan-1-one) with maltose from potato starch; this has been identified after cooking potatoes in microwavable plastic food containers. Additionally, polypropylene glycol substances have been found to transfer into food through microwave cooking. Identifying these substances formed in situ requires state-of-the-art high-resolution mass spectrometry instrumentation and metabolomics-based strategies.

Distribution of chemical residues in the beehive compartments and their transfer to the honeybee brood.

Honeybee (Apis mellifera) is one of the most important crop and wild plant pollinators, playing an essential role in the agricultural production and the natural ecosystems. However, the number of honeybee colonies is decreasing alarmingly, which has motivated extensive research on the factors affecting their development and survival in some regions. Honeybees' exposure to pesticides and other chemicals has been identified as one of the causes of their decline. The present study evaluates the distribution of plant protection products, veterinary treatments and environmental contaminants inside the beehive, their persistence and their migration to the bee brood. During the five-month sampling period, only amitraz was applied to the colonies. Samples of beeswax, beebread (processed pollen) and bee brood were extracted and analyzed using GC-MS/MS and LC-MS/MS with a multiresidue method. The results showed the presence of 31 chemical residues in the samples. The highest concentrations of residues were detected in the beeswax and corresponded to amitraz (expressed as the sum of DMF and DMPF), coumaphos and tau-fluvalinate, with total concentrations of up to 16,858, 7102 and 1775 µg kg-1, respectively. These and other veterinary treatments were found to accumulate in the beeswax and migrate to other beehive matrices such as beebread and bee brood. Plant protection products used in agriculture were also found in the beehive matrices, especially in the beebread. Five different chemical residues (acrinathrin, amitraz, coumaphos, cypermethrin and tau-fluvalinate) were found in bee brood samples at concentration levels ranging from 1 to 167 µg kg-1. These findings reveal that bee brood reared in field conditions is in fact exposed to plant protection products and veterinary residues through direct contact with contaminated wax and via beebread although they had not been applied to the beehive.

Identification of unexpected chemical contaminants in baby food coming from plastic packaging migration by high resolution accurate mass spectrometry.

Plastic multilayers are widely used for baby food packaging. However, it is important to consider that migration of food contact materials (FCM) into the baby food can occur. The comprehensive identification of potential migrants, including intentionally added substances (IAS) and non-intentionally added substances (NIAS), is required to assess the safety of these packaging materials. In this study, high resolution accurate mass spectrometry (HRAMS) with a data-independent acquisition method of sequential mass windows enables the detection of substances with corresponding deconvoluted fragment mass spectra. The identification of unexpected migrants present in the food simulants and in real baby food was facilitated by filtering strategies and by an in-house library. This approach has allowed the identification of 42 migrants, including eight NIAS detected for the first time. Two oligomers were quantified by means of reference standard materials at concentration levels above 0.010 mg/kg, exceeding the maximum residue levels for baby food.

High-resolution mass spectrometry with data independent acquisition for the comprehensive non-targeted analysis of migrating chemicals coming from multilayer plastic packaging materials used for fruit purée and juice.

EU legislation requires that chemicals migrating from food packaging into food do not endanger human health. A proper safety assessment must go further than simply testing for known ingredients used to make the packaging materials. The identification of all potential migrants, including non-intentionally added substances (NIAS), is required to assess the safety of these materials. In this study, the benefits of using LC-ESI-Q-TOF-MS equipment combined with the data-independent acquisition method SWATH (Sequential Windowed Acquisition of All THeoretical MS) have been evaluated for the identification of unexpected migrants from multilayer plastic packaging materials. Identification of non-target peaks followed by filtering strategies facilitated the comprehensive identification procedure of unexpected migrants present in the food simulants. MS and MS/MS data from the most abundant ions in the samples were processed using formula finding and searched against open chemical and spectral databases, such as ChemSpider and METLIN, combined with bibliographic search. This approach has allowed the identification of 26 potential migrants, of which 23 were NIAS. Cyclic oligomers, possibly coming from polyurethane adhesives, were the main migrants in the studied materials. Evaluation of the toxicity of the tentatively identified migrants was carried out, showing that most of them were classified in the higher toxicity class according to Cramer rules. Most of the compounds detected in this study were previously reported in other studies of NIAS migration from plastic packaging materials, except for five of the tentatively identified oligomers that were found for the first time in this study. This study shows the high potential of non-targeted approaches using high resolution accurate mass spectrometry (HRAMS) with SWATH acquisition for the characterization of food packaging materials aimed at food safety evaluation.

Analysis and evaluation of (neuro)peptides in honey bees exposed to pesticides in field conditions.

During the last years, declines in honey bee colonies are being registered worldwide. Cholinergic pesticides and their extensive use have been correlated to the decline of pollinators and there is evidence that pesticides act as neuroendocrine disruptors affecting the metabolism of neuropeptides. However, there is a big absence of studies with quantitative results correlating the effect of pesticide exposure with changes on neuropeptides insects, and most of them are conducted under laboratory conditions, typically with individual active ingredients. In this study, we present an analytical workflow to evaluate pesticide effects on honey bees through the analysis of (neuro)peptides. The workflow consists of a rapid extraction method and liquid chromatography with triple quadrupole for preselected neuropeptides. For non-target analysis, high resolution mass spectrometry, multivariate analysis and automatic identification of discriminated peptides using a specific software and protein sequence databases. The analytical method was applied to the analysis of target and non-target (neuro)peptides in honey bees with low and high content of a wide range of pesticides to which have been exposed in field conditions. Our findings show that the identification frequency of target neuropeptides decreases significantly in honey bees with high concentration of pesticides (pesticide concentrations ≥ 500 µg kg-1) in comparison with the honey bees with low content of pesticides (pesticide concentrations ≤ 20 µg kg-1). Moreover, the principal component analysis in non-target search shows a clear distinction between peptide concentration in honey bees with high level of pesticides and honey bees with low level. The use of high resolution mass spectrometry has allowed the identification of 25 non-redundant peptides responsible for discrimination between the two groups, derived from 18 precursor proteins.

Discovery of novel per- and polyfluoroalkyl substances (PFASs) at a fire fighting training ground and preliminary investigation of their fate and mobility.

Aqueous film forming foams (AFFFs) have been released at fire training facilities for several decades resulting in the contamination of soil and groundwater by per- and polyfluoroalkyl substances (PFASs). AFFF compositions are proprietary and may contain a broad range of PFASs for which the chemical structures and degradation products are not known. In this study, high resolution quadrupole-time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS) in combination with data processing using filtering strategies was applied to characterize and elucidate the PFASs present in concrete extracts collected at a fire training ground after the historical use of various AFFF formulations. Twelve different fluorochemical classes, representing more than 60 chemicals, were detected and identified in the concrete extracts. Novel PFASs homologues, unmonitored before in environmental samples such as chlorinated PFSAs, ketone PFSAs, dichlorinated PFSAs and perfluoroalkane sulphonamides (FASAs) were detected in soil samples collected in the vicinity of the fire training ground. Their detection in the soil cores (from 0 to 2 m) give an insight on the potential mobility of these newly identified PFASs.

Rapid screening and identification of chemical hazards in surface and drinking water using high resolution mass spectrometry and a case-control filter.

Access to clean, safe drinking water poses a serious challenge to regulators, and requires analytical strategies capable of rapid screening and identification of potentially hazardous chemicals, specifically in situations when threats to water quality or security require rapid investigations and potential response. This study describes a fast and efficient chemical hazard screening strategy for characterising trace levels of polar organic contaminants in water matrices, based on liquid chromatography high resolution mass spectrometry with post-acquisition 'case-control' data processing. This method allowed for a rapid response time of less than 24 h for the screening of target, suspect and non-target unknown chemicals via direct injection analysis, and a second, more sensitive analysis option requiring sample pre-concentration. The method was validated by fortifying samples with a range of pesticides, pharmaceuticals and personal care products (n = 46); with >90% of target compounds positively screened in samples at 1 ng mL-1, and 46% at 0.1 ng mL-1 when analysed via direct injection. To simulate a contamination event samples were fortified with compounds not present in the commercial library (designated 'non-target compounds'; fipronil and fenitrothion), tentatively identified at 0.2 and 1 ng mL-1, respectively; and a compound not included in any known commercial library or public database (designated 'unknown' compounds; 8Cl- perfluorooctanesulfonic acid), at 0.8 ng mL-1. The method was applied to two 'real-case' scenarios: (1) the assessment of drinking water safety during a high-profile event in Brisbane, Australia; and (2) to screen treated, re-circulated drinking water and pre-treated (raw) water. The validated workflow was effective for rapid prioritisation and screening of suspect and non-target potential hazards at trace levels, and could be applied to a wide range of matrices and investigations where comparison of organic contaminants between an affected and control site and or timeframe is warranted.

A strategic screening approach to identify transformation products of organic micropollutants formed in natural waters.

Many transformation products (TPs) from organic micropollutants are not included in routine environmental monitoring programs due to limited knowledge of their occurrence and fate. An efficient method to identify and prioritize critical compounds in terms of environmental relevance is needed. In this study, we applied a strategic screening approach based on a case-control concept to identify TPs formed along wastewater-impacted rivers. Time-integrated samples were collected over one week at both ends of a river stretch downstream of a wastewater treatment plant (WWTP) outfall and were analyzed by ultrahigh performance liquid chromatography interfaced with quadrupole time-of-flight mass spectrometry (UHPLC-QToF-MS/MS). The screening procedure of the high-resolution MS (HRMS) datasets consisted of three major steps: (i) screening for parent compounds (PCs) attenuated along the stretch; (ii) prediction of potential TPs from these PCs; and (iii) screening for TPs from this list with an increasing trend along the stretch. In total, 32 PCs decreased along the investigated river stretches. From these PCs, eight TPs had increasing concentrations along the studied stretches and could be tentatively identified. The identification of one TP (benzamide) was confirmed by its corresponding reference standard, while no standards were available for the remaining TPs.

Development of sample extraction and clean-up strategies for target and non-target analysis of environmental contaminants in biological matrices.

Recently, there has been an increasing trend towards multi-targeted analysis and non-target screening methods as a means to increase the number of monitored analytes. Previous studies have developed biomonitoring methods which specifically focus on only a small number of analytes with similar physico-chemical properties. In this paper, we present a simple and rapid multi-residue method for simultaneous extraction of polar and non-polar organic chemicals from biological matrices, containing up to 5% lipid content. Our method combines targeted multi-residue analysis using gas chromatography triple quadrupole mass spectrometry (GC-QqQ-MS/MS) and a multi-targeted analysis complemented with non-target screening using liquid chromatography coupled to a quadrupole time of flight mass spectrometry (LC-QTOF-MS/MS). The optimization of the chemical extraction procedure and the effectiveness of different clean-up methods were evaluated for two biological matrices: fish muscle (lipid content ∼2%) and breast milk (∼4%). To extract a wide range of chemicals, the partition/extraction procedure used for the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) approach was tested as the initial step for the extraction of 77 target compounds covering a broad compound domain. All the target analytes have different physico-chemical properties (log Kow ranges from -0.3 to 10) and cover a broad activity spectrum; from polar pesticides, pharmaceuticals, personal care products (PPCPs) to highly lipophilic chemicals such as polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) and organochloride pesticides (OCPs). A number of options were explored for the clean-up of lipids, proteins and other impurities present in the matrix. Zirconium dioxide-based sorbents as dispersive solid-phase extraction (d-SPE) and protein-lipid removal filter cartridges (Captiva ND Lipids) provided the best results for GC-MS and LC-MS analysis respectively. The method was fully validated for samples of fish muscle and breast milk through the evaluation of recoveries, matrix effects, limit of quantification, linearity and precision (inter-day and intra-day). Mean recoveries (n=5) were between 70 and 120% with relative standard deviations (RSD) less than 20% in most of the cases. GC-MS/MS LOQs ranged from 0.08 to 3µg/kg and LC-QTOF-MS/MS LOQs ranged from 0.2 to 9µg/kg. The developed strategy was successfully applied for analysis of real samples; 22 target analytes were found in the breast milk samples and 10 in the fish samples. Non-target analysis allowed the detection and identification of an additional 14 contaminants and metabolites in the samples.

Novel fluorinated surfactants tentatively identified in firefighters using liquid chromatography quadrupole time-of-flight tandem mass spectrometry and a case-control approach.

Fluorinated surfactant-based aqueous film-forming foams (AFFFs) are made up of per- and polyfluorinated alkyl substances (PFAS) and are used to extinguish fires involving highly flammable liquids. The use of perfluorooctanesulfonic acid (PFOS) and other perfluoroalkyl acids (PFAAs) in some AFFF formulations has been linked to substantial environmental contamination. Recent studies have identified a large number of novel and infrequently reported fluorinated surfactants in different AFFF formulations. In this study, a strategy based on a case-control approach using quadrupole time-of-flight tandem mass spectrometry (QTOF-MS/MS) and advanced statistical methods has been used to extract and identify known and unknown PFAS in human serum associated with AFFF-exposed firefighters. Two target sulfonic acids [PFOS and perfluorohexanesulfonic acid (PFHxS)], three non-target acids [perfluoropentanesulfonic acid (PFPeS), perfluoroheptanesulfonic acid (PFHpS), and perfluorononanesulfonic acid (PFNS)], and four unknown sulfonic acids (Cl-PFOS, ketone-PFOS, ether-PFHxS, and Cl-PFHxS) were exclusively or significantly more frequently detected at higher levels in firefighters compared to controls. The application of this strategy has allowed for identification of previously unreported fluorinated chemicals in a timely and cost-efficient way.

Comprehensive two-dimensional GC of supercritical fluid and pressurized liquid extracts of tobaccos.

The developments of an analytical strategy based on rapid extraction techniques coupled to comprehensive bi-dimensional gas chromatography (GCxGC) for the characterization of the volatile fraction of tobaccos are presented. The high-peak capacity of GCxGC allows considering global extraction techniques that do not focus on restricted chemical families of products. Thus, potential of pressurized fluid extraction and supercritical fluid extraction (SFE) to provide informative extracts is evaluated and compared. As expected, both techniques enable rapid extraction of compounds of interest. However, because of the easy removal of the extracting fluid, SFE provides more concentrated extracts, allowing a GCxGC direct injection without any concentration step. Moreover, the use of pure carbon dioxide with tuneable extraction power favors the extraction of the compounds of interest while reducing the extraction of interfering heavy compounds. GCxGC conditions, especially concerning the set of columns used, are selected thanks to the comparison of separations obtained on model compounds. The developed methodology is applied not only to compare the three main types of tobaccos but also to discriminate between different tobaccos of the same type.