From sound check to encore: A journey through high-resolution mass spectrometry-based food analyses and metabolomics.

This manuscript presents a comprehensive review of high-resolution mass spectrometry in the field of food analysis and metabolomics. We have followed the historical evolution of metabolomics, its associated techniques and technologies, and its increasing role in food science and research. The review provides a critical comparison and synthesis of tentative identification guidelines proposed for over 15 years, offering a condensed resource for researchers in the field. We have also examined a wide range of recent metabolomics studies, showcasing various methodologies and highlighting key findings as a testimony of the versatility of the field and the possibilities it offers. In doing so, we have also carefully provided a compilation of the software tools that may be employed in this type of studies. The manuscript also explores the prospects of high-resolution mass spectrometry and metabolomics in food science. By covering the history, guidelines, applications, and tools of metabolomics, this review attempts to become a comprehensive guide for researchers in a rapidly evolving field.

Improved efficiency of ion trapping time-of-flight mass spectrometry for the analysis of pesticide residues and mycotoxins at trace levels in baby food.

The analysis of pesticide residues and mycotoxins in baby food demands exceptionally low limits of quantitation, necessitating the use of highly sensitive instruments capable of conducting trace analyses. High-resolution instruments typically fail to detect such low levels. However, the latest advancements in liquid time-of-flight technology, when coupled with ion trapping, enable ion enrichment, thereby improving detection levels. This allows for the analysis of these substances at low concentration levels, benefiting from enhanced mass accuracy. Additionally, the use of mass accuracy data helped eliminate matrix interferences, thereby enabling high-confidence identification. We developed a multi-residue method to analyse 219 pesticide residues and 9 mycotoxin residues in baby food matrices. Utilizing a QuEChERS-based extraction method, the samples were then analysed using an LC-Zeno® trap QTOF with mass window screening acquisition. For pesticides, the limit of quantitation was 0.001-0.003 mg/kg for 81 % of the evaluated compounds, 0.005 mg/kg for 13 %, 0.010 mg/kg for 4 % and 0.020-0.030 for 2 %; good linearities were obtained at these levels. Apparent recoveries were evaluated at 0.003, 0.005, and 0.010 mg/kg. At the lowest recovery level, 93 % of compounds showed recoveries between 70 and 120 %. The rest of the compounds were in the range of 63-129 %, with relative standard deviation values below 20 %. For mycotoxins, the limits of quantitation ranged from 0.0001 to 0.100 mg/kg, with matrix-matched concentrations assessed within this range. Recoveries were evaluated at low concentration range (0.001-0.003 mg/kg) and high range (0.020-0.050) with apparent recoveries values between 92 and 140 %. Finally, a total of 31 commercial baby food samples were analysed using this method. The results indicated that 16 samples contained pesticide residues, while two samples were found to have mycotoxins.

Collision cross-section as a universal molecular descriptor in the analysis of PFAS and use of ion mobility spectrum filtering for improved analytical sensitivities.

The massive usage of per- and polyfluoroalkyl substances (PFAS), as well as their high chemical stability, have led to their ubiquitous presence in environmental matrices and direct human exposure through contaminated food, particularly fish. In the analysis of this large group of substances, the use of ion mobility coupled to mass spectrometry is of particular relevance because it uses an additional descriptor, the collision cross-section (CCS), which results in increased selectivity. In the present work, the TWCCSN2 of 24 priority PFAS were experimentally obtained, and the reproducibility of these measurements was evaluated over seven weeks. The average values were employed to critically assess previously reported data and theoretical calculations. This gain in selectivity made it possible to increase the sensitivity of the detection on complex matrices (biota, food and human serum) by using the drift time associated to each analyte as a filter, thus reducing the interferences and background noise and allowing their detection at trace levels.

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.

Cutting-edge approach using dual-channel chromatography to overcome the sensitivity issues associated with polarity switching in pesticide residues analysis.

A fast and sensitive method was validated for the analysis of pesticide residues in baby food. After an QuEChERS-based extraction, the samples were analysed with a dual-channel liquid chromatography instrument coupled to a triple quadrupole mass spectrometer. The method consisted of two independent injections per sample. In the first injection the sample was analysed with a mobile phase optimal for the positive polarity ionisation (water, methanol, formic acid, and ammonium formate), whereas in the second injection the mobile phase was optimised for the negative polarity ionisation (water, acetonitrile, acetic acid). The total number of pesticides was 264, out of which 238 were analysed with the methanol gradient and 26 with the acetonitrile gradient. The dual-channel instrument allowed for sample multiplexing. Thus, sample throughput was equivalent to that of a single-channel system. The limit of quantitation, determined according to the DG SANTE guidance document, was 0.003 mg/kg for 97% of the evaluated compounds. The validation study was followed by a real sample survey. In 42 samples, 16 positives were found.

Improving the simultaneous target and non-target analysis LC-amenable pesticide residues using high speed Orbitrap mass spectrometry with combined multiple acquisition modes.

The use of high-resolution mass spectrometry (HRMS) for the simultaneous target and non-target analysis of pesticide residues in food control is a subject that has been studied over the last decade. However, proving its efficacy compared to the more established triple quadrupole mass spectrometers (QQQ-MS2) is challenging. Various HRMS platforms have been evaluated, seemingly showing this approach not to be as effective as QQQ-MS2 for quantitative analysis, especially in routine food testing laboratories. The two main reasons are (i) the lower sensitivity especially in the case of the fragment ions produced and (ii) the lack of familiarity and an understanding of the most appropriate combination of HRMS acquisition modes to use. In fact, the number of different acquisition modes can appear as a puzzle to inexperienced users. This work was therefore focused on obtaining experimental data to gain a better understanding of the extended acquisition capabilities of a new Q-Orbitrap platform. Experimental data were obtained for 244 pesticides and their degradation products in commodities of varying matrix complexity (tomato, onion, avocado, and orange) using various combinations of acquisition modes. The best results for targeted analysis were obtained with a combination of full scan (FS), all-ions fragmentation (AIF) and target MS2 (tMS2) modes, and for non-target analysis using full scan (FS) and data-dependent MS2 (ddMS2) modes. All these acquisition modes (FS, AIF, tMS2, and ddMS2) could be applied simultaneously with cycle times ≤ 1 s. The tMS2 especially, proved to be a very powerful approach to increase sensitivity for MS2 fragments and identification rates. Overall, the results for the various pesticide-commodity combinations were fully satisfactory in terms of limit of quantitation (LOQ) repeatability and identification when considered against the SANTE EU Guideline criteria. In addition, the screening capabilities were evaluated for a non-target survey with the use of spectral libraries, the presence of non-target compounds was detected, thus proving the efficacy of the proposed approach. Another issue often overlooked is the optimization of use of spectral libraries, but in our experiments the compounds present in these libraries were not blindly sought in the screening analyses. To minimize the potential for false positives detects in our study, the extractability of the compounds present in the libraries, was also taken into account. The extractability of compounds using a QuEChERS acetonitrile procedure was estimated based on the physicochemical properties of target compounds. By removing compounds that will not be extracted, reduces the occurrences of false detects, reducing the time required for data processing and thus improving the efficiency of the overall screening workflow.

Validation of a quick and easy extraction method for the determination of emerging contaminants and pesticide residues in agricultural soils.

Treated wastewater is currently used in the agricultural sector to solve the lack of availability of freshwater in many regions. However, reclaimed water can contain multiclass of organic contaminants. Therefore, the soil can become a reservoir of agricultural (e.g. pesticides) and urban (e.g. pharmaceuticals) contaminants. Consequently, the evaluation of this contamination process is necessary for assessing its potential human and environmental negative effects. Due to the low concentration levels, different chemical properties and the complexity of the matrix, an efficient sample preparation step for achieving adequate sensitivity and robust analysis in the soil is needed. The aim of this study was to develop a quick and easy extraction method based on a QuEChERS procedure for the determination of 27 organic contaminants in agricultural soil samples. The procedure was based on a salting-out extraction with acidified acetonitrile, followed by a dispersive solid-phase extraction (d-SPE). A liquid chromatography-tandem mass-spectrometry (LC-MS/MS) system was applied for the determination and quantification of the selected target analytes. The main benefits of this analytical approach are:•Reduction/elimination of majority of the interferences improving the sensitivity of the method.•Robust simultaneous determination of a multiclass of organic contaminants with very different physicochemical properties.

Environmental monitoring study of pesticide contamination in Denmark through honey bee colonies using APIStrip-based sampling.

Due to their extensive use in both agricultural and non-agricultural applications, pesticides are a major source of environmental contamination. Honey bee colonies are proven sentinels of these and other contaminants, as they come into contact with them during their foraging activities. However, active sampling strategies involve a negative impact on these organisms and, in most cases, the need of analyzing multiple heterogeneous matrices. Conversely, the APIStrip-based passive sampling is innocuous for the bees and allows for long-term monitorings using the same colony. The versatility of the sorbent Tenax, included in the APIStrip composition, ensures that comprehensive information regarding the contaminants inside the beehive will be obtained in one single matrix. In the present study, 180 APIStrips were placed in nine apiaries distributed in Denmark throughout a six-month sampling period (10 subsequent samplings, April to September 2020). Seventy-five pesticide residues were detected (out of a 428-pesticide scope), boscalid and azoxystrobin being the most frequently detected compounds. There were significant variations in the findings of the sampling sites in terms of number of detections, pesticide diversity and average concentration. A relative indicator of the potential risk of pesticide exposure for the honey bees was calculated for each sampling site. The evolution of pesticide detections over the sampling periods, as well as the individual tendencies of selected pesticides, is herein described. The findings of this large-scale monitoring were compared to the ones obtained in a previous Danish, APIStrip-based pilot monitoring program in 2019. Samples of honey and wax were also analyzed and compared to the APIStrip findings.

Presence of anthraquinone in coffee and tea samples. An improved methodology based on mass spectrometry and a pilot monitoring programme.

Anthraquinone has been linked to potential adverse effects on human health and the environment. The most commonly employed methods for the analysis of coffee and tea cause the extraction of matrix interferents such as the methylxanthines caffeine and theobromine, which hinder the analysis of anthraquinone. A new manual extraction method - using ethyl acetate as the extraction solvent with a dispersive solid-phase extraction clean-up step based on primary-secondary amines - has been developed. The new developed method allows for the quantitation of anthraquinone at 5 µg kg-1 concentration levels, four times lower than the current maximum residue limit for coffee and tea in the European Union (20 µg kg-1). Alongside, a new automated extraction method has also been developed. Finally, a pilot monitoring programme of 90 coffee and tea samples from several countries within the European Union has been performed, in which anthraquinone has been detected in a concentration range of 5.1-18.8 µg kg-1 in 32% of the monitored samples, below the current 20 µg kg-1 maximum residue limit, and in 48% of the monitored tea samples, revealing the need for including anthraquinone in a more extensive monitoring programme of tea.

Dissipation and cross-contamination of miticides in apiculture. Evaluation by APIStrip-based sampling.

The active substances coumaphos, tau-fluvalinate and amitraz are among the most commonly employed synthetic miticides to control varroa infestations in apiculture. These compounds can persist inside the beehive matrices and can be detected long time after their application. The present study describes the application of a new passive sampling methodology to assess the dissipation of these miticides as well as the cross-contamination in neighboring beehives. The APIStrips are a recently developed sampling device based on the sorbent Tenax, which shows a remarkable versatility for the sorption of molecules onto its surface. This avoids the need of actively sampling apicultural matrices such as living bees, wax or reserves (honey and pollen), therefore allowing to obtain representative information of the contamination in the beehive environment in one single matrix. The results show that the amitraz-based treatments have the fastest dissipation rate (half-life of 11-14 days), whereas tau-fluvalinate and coumaphos remain inside the beehive environment for longer time periods, with a half-life up to 39 days. In the present study, tau-fluvalinate originated an intense cross-contamination, as opposed to coumaphos and amitraz. This study also demonstrates the contribution of drifting forager bees in the pesticide cross-contamination phenomena. Moreover, the sampling of adult living bees has been compared to the APIStrip-based sampling, and the experimental results show that the latter is more effective and consistent than traditional active sampling strategies. The active substances included in this study do not migrate to the honey from the treated colonies in significant amounts.

Dual-channel chromatography a smart way to improve the analysis efficiency in liquid chromatography coupled to mass spectrometry.

Dual-channel chromatography was evaluated for pesticide residue analysis in fruits and vegetables and for unknown compounds detection. A dual-channel system was tested coupled to triple quadrupole and high-resolution mass spectrometry. The first part of the investigation was related to the improvement of the sample throughput with a 100 mm column. The dual-channel system provided the same analytical results as the single-channel system, however, with the throughput higher of about 70% (80 injections vs 137 injections in 24 h). Two types of calibration (in-channel and cross-channel) were checked. In the article, also solvent consumption is discussed. Six proficiency test samples were analysed to assess the quality of the results. Nor false positives neither false negatives were found. Calculated z-scores were typically <1. In the second part, a different approach was investigated. The 100 mm column was replaced by a 150 mm column keeping shorter run times than single channel system and 100 mm. The longer column improved the sensitivity and selectivity what was demonstrated in the target pesticide residue analysis. Additionally, the 150 mm column was compared with the 100 mm column in the analysis of unknown natural matrix compounds by high resolution mass spectrometry. The longer column allowed to detect up to 26% unknown compounds more than the shorter column.

Supercritical fluid chromatography separation of chiral pesticides: Unique capabilities to study cyhalothrin and metalaxyl as examples.

Evaluation of chiral pesticides remains a frequently neglected matter in routine food control laboratories. This fact is due to the existence of many residue definitions but also due to the lack of robust instrumental methods for the evaluation of these isomeric compounds. However, supercritical fluid chromatography coupled to mass spectrometry (SFC-ESI-MS/MS) has been demonstrated to perform fast and highly efficient separations without the need to change the mobile phase employed in multiresidue pesticide analyses. Regarding chiral stationary phase columns, the polysaccharide-based ones clearly demonstrate the best separation technology. Two polysaccharide-based columns were tested in this study, and the robustness of their combination with SFC was verified. The enantiomers of lambda-cyhalothrin and metalaxyl were studied precisely due to their markedly distinct toxicity and enantioselectivity. Furthermore, the acute reference dose for gamma-cyhalothrin is half in comparison with its enantiomer (0.0025 and 0.005 mg/kg respectively), which is present in the lambda-cyhalothin residue definition. These enantiomers were analyzed in terms of linearity, reproducibility, and matrix effects in four representative matrices (tomato, orange, leek, and cayenne). Additionally, field tests under greenhouse conditions for these compounds were performed. The results obtained after different sample collections revealed a similar degradation in lambda-cyhalothrin enantiomers (R, S, S, and S, R, R) but not in the case of metalaxyl-M (mefenoxam) where the degradation in tomato was 2 to 6 times less in comparison with its S-enantiomer.

APIStrip, a new tool for environmental contaminant sampling through honeybee colonies.

Honeybee colonies are proven bio-samplers in their foraging area, as organic contaminants such as pesticides are continuously deposited in their hives. However, the use of honeybee colonies for the biomonitoring of contaminants requires the sampling of biological matrices such as bees, pollen, honey or beeswax. This active sampling alters the colonies, especially in the case of frequent sampling intervals. In this study, a non-biological passive sampler based on Tenax TA is described: the APIStrip (Adsorb Pesticide In-hive Strip). A concentrated solution of Tenax in dichloromethane has been applied to a polystyrene strip, resulting in a bee-proof, in-hive passive sampler. The pesticides and related contaminants adsorbed onto its surface can be extracted in acetonitrile and analyzed by LC-MS/MS and GC-MS/MS. The APIStrip preparation has been optimized, the optimal exposure period has been stablished as 14 days and the stability of the pesticides on the APIStrip surface has been evaluated. Preliminary tests demonstrated the efficacy, sensitivity, representativeness and reproducibility of the APIStrip-based sampling when compared to the analysis of beeswax comb, which facilitates the detection of contaminants even in beehives exposed to low polluting pressure. Field studies in Denmark, performed in the INSIGNIA monitoring study over a six-month period, demonstrated their value and applicability by detecting 40 different pesticide residues.

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.

A non-targeted metabolomic approach to identify food markers to support discrimination between organic and conventional tomato crops.

In the last decade, the consumption trend of organic food has increased dramatically worldwide. However, the lack of reliable chemical markers to discriminate between organic and conventional products makes this market susceptible to food fraud in products labeled as "organic". Metabolomic fingerprinting approach has been demonstrated as the best option for a full characterization of metabolome occurring in plants, since their pattern may reflect the impact of both endogenous and exogenous factors. In the present study, advanced technologies based on high performance liquid chromatography-high-resolution accurate mass spectrometry (HPLC-HRAMS) has been used for marker search in organic and conventional tomatoes grown in greenhouse under controlled agronomic conditions. The screening of unknown compounds comprised the retrospective analysis of all tomato samples throughout the studied period and data processing using databases (mzCloud, ChemSpider and PubChem). In addition, stable nitrogen isotope analysis (δ15N) was assessed as a possible indicator to support discrimination between both production systems using crop/fertilizer correlations. Pesticide residue analyses were also applied as a well-established way to evaluate the organic production. Finally, the evaluation by combined chemometric analysis of high-resolution accurate mass spectrometry (HRAMS) and δ15N data provided a robust classification model in accordance with the agricultural practices. Principal component analysis (PCA) showed a sample clustering according to farming systems and significant differences in the sample profile was observed for six bioactive components (L-tyrosyl-L-isoleucyl-L-threonyl-L-threonine, trilobatin, phloridzin, tomatine, phloretin and echinenone).

Coupling Ion Chromatography to Q-Orbitrap for the Fast and Robust Analysis of Anionic Pesticides in Fruits and Vegetables.

Ion chromatography coupled to a quadrupole Orbitrap mass analyzer was used to develop a multiresidue method for the determination of highly polar pesticides and their metabolites (chlorate, perchlorate, fosetyl-aluminum, glyphosate, aminomethylphosphonic acid (AMPA), phosphonic acid, N-acetyl AMPA, and N-acetyl glyphosate) in fruits and vegetables. After extraction with methanol, samples were diluted 5× with water. No derivatization was applied. Pesticides were separated in an anion-exchange column. Water was used as the ion chromatography mobile phase. A gradient was created by increasing the concentration of KOH in the mobile phase. Ion chromatography provided good and stable retention and separation for all studied compounds. All investigated pesticides had an LOQ of 0.01 mg/kg and a linear range of 0.01-0.50 mg/kg. The ion ratio of the m/z ions produced was stable and adequate (deviation <30%) in all cases. The obtained mass errors (always in full-scan MS and MS2 mode) were <0.2 mDa. The high resolution (>100 000) provided by the Orbitrap analyzer with the low m/z ions obtained (e.g., m/z 80) was effective in obtaining low background matrix signals. The influence of postcolumn infusion of organic solvent on sensitivity was investigated. Acetonitrile was found to be more effective than methanol, increasing the sensitivity 3× with respect to water. The method was validated for five vegetable-based matrixes. Both the sample processing and the analytical measurement were very fast. Hence, the methodology is ideal for high-throughput work.