Comprehensive analysis of contaminants in Brazilian infant formulas: Application of QuEChERS coupled with UHPLC-QqQ-MS/MS and suspect screening-unknown analysis by UHPLC-Q-Orbitrap-MS.

Infant formulas (IF) can contain harmful chemical substances, such as pesticides and mycotoxins, resulting from the contamination of raw materials and inputs used in the production chain, which can cause adverse effects to infants. Therefore, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) methodology prior ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPL-QqQ-MS/MS) analysis was applied for the determination of 23 contaminants, in 30 samples of Brazilian IF. The method was validated in terms of limit of detection (0.2 to 0.4 µg/kg), limits of quantification (1 and 10 µg/kg), and recovery (64 % to 122 %); precision values, in terms of relative standard deviation (RSD), were ≤ 20 %. Fenitrothion, chlorpyrifos, and bifenthrin were the pesticides detected in the samples, but the values did not exceed the limit set by the European Union (EU), and ANVISA, and they were detected under their limits of quantification. Additionally, suspect screening and unknown analysis were conducted to tentatively identify 32 substances, including some compounds not covered in this study, such as pesticides, hormones, and veterinary drugs. Carbofuran was identified, confirmed and quantified in 10 % of the samples.

Development and Application of a Novel Pluri-Residue Method to Determine Polar Pesticides in Fruits and Vegetables through Liquid Chromatography High Resolution Mass Spectrometry.

Nowadays, highly polar pesticides are not included in multiresidue methods due to their physico-chemical characteristics and therefore, specific analytical methodologies are required for their analysis. Laboratories are still looking for a pluri-residue method that encompasses the largest number of polar pesticides. The aim of this work was the simultaneous determination of ethephon, 2-hydroxyethylphosphonic acid (HEPA), fosetyl aluminum, glyphosate, aminomethylphosphonic acid (AMPA), N-acetyl-glyphosate and N-acetyl-AMPA in tomatoes, oranges, aubergines and grapes. For that purpose, an ultra high performance liquid chromatography (UHPLC) coupled to a high resolution single mass spectrometer Orbitrap-MS were used. Different stationary phases were evaluated for chromatographic separation, and among them, the stationary phase Torus DEA provided the best separation of the selected compounds. The QuPPe method was used for the extraction of the analytes, but slight modifications were needed depending on the matrix. The developed method was validated, observing matrix effect in all matrices. Intra- and inter-day precision were estimated, and relative standard deviation were lower than 19%. Recoveries were satisfactory, and mean values ranged from 70% to 110%. Limits of quantification were between 25 and 100 µg kg-1. Finally, the analytical method was applied to different fruits and vegetables (oranges, tomatoes, aubergines and grapes).

Application of full scan gas chromatography high resolution mass spectrometry data to quantify targeted-pesticide residues and to screen for additional substances of concern in fresh-food commodities.

A novel application, which combines targeted and non-targeted approaches, using gas chromatography high-resolution mass spectrometry (GCHRMS) for multiresidue analysis of pesticides in fruits and vegetables is proposed; the aim is to add information to that obtained from targeted methods to be used in pesticide residues monitoring laboratories. Extract from fruit samples obtained with QuEChERS method are analysed with two different approaches: a first step in targeted mode in full scan, and a second step in non-targeted mode with HRMS deconvolution. The targeted method was validated for 191 pesticides, in banana, watermelon, pear and strawberry, with recoveries within 70-120%, precision-RSD <20%, linearity between 1-100 µg/kg (5-100 µg/kg in strawberry) and quantification limits (LOQs) of 5 µg/kg. For targeted method, 12 different pesticides in 14 samples were detected. The non-targeted approach allowed the corroboration of all the results obtained through targeted method, in addition other compounds were tentatively identified.

Evaluation of the behaviour of propoxycarbazone herbicide in soils and water under different conditions. Post-targeted study.

The aim of this study is to understand the levels and behaviour of the herbicide propoxycarbazone in environmental compartments, which are poorly characterized considering degradation products that might reach either surface water as groundwater or soil samples. To this end, an analytical method, based on ultra-high performance liquid chromatography (UHPLC) coupled with Orbitrap mass spectrometry (MS), has been developed to determine the parent compound and its transformation products in soils and water. In addition, a set of laboratory trials has been designed to address the dissipation process of propoxycarbazone formulation in both environmental substrates. The study revealed that the dissipation in water followed a first order kinetics and a non-linear "First Order Multi Compartment" model (FOMC) to soil. Relevant advances have been also achieved in terms of the analytical methodology, as far as the UHPLC-Orbitrap-MS technique allows the development of a retrospective screening of pesticide transformation products in water and soils. For this purpose, an accurate-mass database has been built and used for the post-run screening analysis with 9 transformation products. Photodegradation presumably plays an important role in the dissipation of propoxycarbazone. The results of UHPLC-Orbitrap-MS revealed that four transformation products were formed during the degradation process of the herbicide.

Determination of Calystegines in Several Tomato Varieties Based on GC-Q-Orbitrap Analysis and Their Classification by ANOVA.

In this study, several calystegines (A3, A5, B1, B2, B3, B4, and C1) were determined in tomato. A simple extraction followed by a derivatization step with silylating agents was performed prior to their analysis by gas chromatography coupled to high resolution mass spectrometry (GC-HRMS-Q-Orbitrap), which allowed the monitoring of several ions at accurate mass. The validation of the method has provided suitable values of linearity, trueness (73.7-120.0%), and precision (≤20.0%, except for calystegines B3 and B4 at 0.5 mg/kg). The limit of quantitation was set at 0.5 mg/kg for all analytes. The validated method was successfully applied to the analysis of nine different tomato varieties, and calystegines A3, A5, B2, and C1 were found at concentrations ranging between 0.65 mg/kg (C1) and 12.47 mg/kg (B2). Tomato varieties were classified according to their calystegines content by applying an analysis of variance (ANOVA).

Automated Determination of Xenobiotics (Pesticides, PCBs, PAHs, and PBDEs) in Sediment Samples Applying HS-SPME-GC-HRMS.

Background: Automated methods are needed for the reliable determination of xenobiotics in environmental samples. Objective: Optimization and application of an automated method for the ultra-trace analysis of 34 organic contaminants including polycyclic aromatic hydrocarbons (PAHs), polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyls (PCBs), organochlorinated (OCPs), and organophosphorus pesticides (OPPs) in sediment samples have been performed. Methods: Automated method based on headspace solid-phase microextraction (SPME) coupled to GC-high-resolution MS (GC-HRMS) for the ultra-trace analysis of the targeted compounds has been developed. Conclusions: Suitable validation parameters in terms of linearity, trueness, selectivity, intraday and interday precision, LODs, and LOQs were obtained. Relative recovery values between 70 and 120% were achieved for all compounds (concentration levels assayed 1 and 10 µg/kg). RSD values were always lower than 25% for intra- and interday precision, and LODs and LOQs were 0.1 and 1.0 µg/kg, respectively, for all analytes. Highlights: The proposed method was applied to the analysis of sediments collected in Andalusia, Spain, and Poland, finding PCB 18 in one sample (15.9 µg/kg) and p,p'-DDE in several samples at concentrations ranging from 27.6 to 297.2 µg/kg.

Automated and simultaneous determination of priority substances and polychlorinated biphenyls in wastewater using headspace solid phase microextraction and high resolution mass spectrometry.

The re-use of wastewater has been in growing demand but a possible negative feature is the possibility of organic pollutants being present after its treatment. In order to control the presence of contaminants in wastewater, a completely automated methodology capable of determining a total of 55 organic pollutants including pesticides, polycyclic aromatic hydrocarbons (PAHs), brominated diphenyl ethers (BDEs) and polychlorinated biphenyls (PCBs) at ultra-trace levels has been developed. The proposed method is based on an on-line combination of headspace solid phase microextraction (HS-SPME) and gas chromatography coupled to double-focusing magnetic sector high resolution mass spectrometry (GC-HRMS). HS-SPME as well as GC-HRMS conditions were optimized so as to achieve maximum extraction efficiency and sensitivity which was reinforced by using multiple ion detection (MID) as acquisition mode. Special attention was directed to fiber conditioning and stability by calorimetric and scanning electron microscopy (SEM) analysis. Method validation showed good linearity (R2 > 0.99), recoveries between 86 and 113%, relative standard deviation (RSD) values < 20% for intra-day and inter-day precision and quantification limits (LOQs) between 0.01 and 350 ng L-1. Finally, the proposed method was successfully applied to wastewater samples revealing the presence of PAHs and PCBsS and certain pesticides, mainly anthracene, chlorpyrifos, p,p'-DDD and p,p'-DDT and the metabolite p,p'-DDE.

Determination of rodenticides and related metabolites in rabbit liver and biological matrices by liquid chromatography coupled to Orbitrap high resolution mass spectrometry.

An analytical method based on ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap high resolution mass spectrometry was developed for the determination of rodenticides (bromadiolone, brodifacoum, difenacoum, chlorophacinone, diphacinone, coumachlor and warfarin) in liver matrix. Different extraction conditions were tested, obtaining the best results when the "dilute and shoot" method (acidified acetonitrile as extraction solvent) and a clean-up step with primary secondary amine (PSA) were used. The optimized method was validated, obtaining recoveries ranging from 60 to 120%. Repeatability and reproducibility were evaluated obtaining values lower than 20%, except for brodifacoum at 10µg/kg. Limits of quantification (LOQs) ranged from 0.1 to 0.5µg/kg, except for brodifacoum, which was 100µg/kg. Six liver samples were analyzed and diphacinone and chlorophacinone were detected in three samples at concentrations ranging from 4µg/kg to 13µg/kg. Moreover a retrospective screening of rodenticide metabolites in those samples and in animal forensic samples was developed based on Orbitrap capabilities. Brodifacoum was detected in three samples, and warfarin alcohol, which is a metabolite of warfarin, was also detected in one sample.

Simple and quick determination of analgesics and other contaminants of emerging concern in environmental waters by on-line solid phase extraction coupled to liquid chromatography-tandem mass spectrometry.

A simple and quick analytical method has been developed for the determination of pharmaceutical compounds in water. An on-line solid-phase extraction (SPE) coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been optimized to determine 7 contaminants of emerging concern in environmental waters at ngL(-1) levels. This procedure requires minimal sample handling and small sample volume (900µL) with a total running time of 18min. Several SPE parameters were evaluated and optimized in order to achieve a high sample throughput. Therefore sample volume, carryover and reusability of the cartridges were evaluated. Performance characteristics were evaluated and good linearity was obtained (R(2)>0.98). Recoveries were evaluated in spiked samples at three concentrations and the values ranged from 71 to 104%. Intra and inter-day precision was lower than 10 and 13% respectively. Limits of quantification were equal to or lower than 10ngL(-1), except for 1,7-dimethylxanthine (20ngL(-1)) and ibuprofen (50ngL(-1)). The method was applied to 20 environmental water samples, and ibuprofen was the compound most widely detected at concentrations up to 42.06µgL(-1), whereas the other compounds were detected in fewer samples at lower concentrations (up to 15.99µgL(-1)).

Influence of storage conditions in the evolution of phytochemicals in nutraceutical products applying high resolution mass spectrometry.

An adequate knowledge about possible transformation of bioactive compounds in nutraceutical products during long storage is important in order to know potential modifications of this type of compounds. In this study, one year monitoring was performed in different types of nutraceutical products based on natural extracts (green tea, soy, royal jelly and grapes) observing the appearance of new bioactive compounds, which were not detected at the initial conditions, as well as the decrease of some of the detected compounds. To determine these transformation products, an analytical procedure based on high resolution mass spectrometry (Exactive-Orbitrap analyzer) was applied. It was noted that transformation products were detected after 3 months of storage in green tea and soy products, while 6 months were necessary to observe transformation products in royal jelly.

Quality control evaluation of nutraceutical products from Ginkgo biloba using liquid chromatography coupled to high resolution mass spectrometry.

Analysis of 11 commercial nutraceutical products obtained from ginkgo has been performed using ultra-high performance liquid chromatography coupled to single-stage Orbitrap high resolution mass spectrometry (UHPLC-Orbitrap-MS). The main phytochemicals present in these samples were detected and quantified, utilizing a database containing 65 compounds. Phytochemicals were extracted using a mixture of an aqueous solution of methanol:water (80:20, v/v) in two sequential solid-liquid extractions. Adequate validation parameters were obtained. The validated compounds exhibited suitable linearity with determination coefficients (R(2)) higher than 0.99, and intra and inter-day precision were lower than 17 and 22%, respectively. Limits of detection (LODs) and quantification (LOQs) were calculated, ranging from 2 to 10 µg L(-1), except for myricetin (LOD, 150 µg L(-1) and LOQ, 300 µg L(-1)). Results indicate that the amount of terpenoids greatly varies among samples, ranging from 1133 (C7) to 12706 mg kg(-1) (C11). This emphasizes the importance of improve quality control in ginkgo-based products. Moreover, retrospective analysis allowed the detection of some undesirable substances as ginkgolic acid in the samples evaluated.

Determination of polycyclic aromatic hydrocarbons in soy isoflavone nutraceutical products by gas chromatography coupled to triple quadrupole tandem mass spectrometry.

Thirteen polycyclic aromatic hydrocarbons have been determined in soy-based nutraceutical products. First, an optimization of extraction procedure was performed, and a solid-liquid extraction assisted by sonication and a dilute and shoot procedure were compared, selecting the dilute and shoot approach for the extraction of target compounds, utilizing a mixture of acetone/n-hexane (1:1 v/v) as extractant solvent. After this, a clean-up step was needed bearing in mind the complexity of these matrices. Dispersive solid-phase extraction, using a mixture of C18 and Zr-Sep+ (25 mg/mL each) was used. The separation was achieved by gas chromatography and detection with triple quadrupole tandem mass spectrometry. For quantification purposes, matrix-matched calibration was used. The validation was applied at three concentration levels (20, 100 and 250 µg/kg), obtaining recoveries between 70 and 120% and precision values equal to or lower than 23%. Limits of detection and quantification were below 8 and 20 µg/kg, respectively. The method was applied in 11 samples, detecting five polycyclic aromatic hydrocarbons at concentrations ranging from 4.1 to 18.5 µg/kg.

Sample Treatment in Pesticide Residue Determination in Food by High-Resolution Mass Spectrometry: Are Generic Extraction Methods the End of the Road?

The use of high-resolution MS (HRMS) is becoming popular in laboratories for the determination of pesticide residues in food commodities. The recent improvements in the instrumentation have helped to increase the number of active compounds and transformation products that can be monitored within a simple chromatographic run. However, prior to instrumental determination, it is necessary to perform a nonspecific, or generic, sample treatment that allows the efficient extraction of several compounds with very relevant differences in their physical and chemical properties. In this sense, the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method and its several modifications and the "dilute-and-shoot" extraction methodology have already revealed an enormous potential for their use together with chromatographic techniques coupled to HRMS. The potentiality and limitations of such a methodological combination have been evaluated in terms of sensitivity and selectivity when they are applied to the analysis of complex food matrixes. An evaluation of the scope of the methods, in terms of efficiency of the extraction and ionization steps, as well as the matrix effect, has also been carried out. Different solutions for the matrix effect have been considered, including improvement in clean-up steps, sample dilution, and matrix compensation by matrix-matched calibration or by the use of isotopically labeled standards.

Development and Validation of a Multiresidue Method for the Determination of Pesticides in Dry Samples (Rice and Wheat Flour) Using Liquid Chromatography/Triple Quadrupole Tandem Mass Spectrometry.

A rapid and sensitive multiresidue method was developed and validated for the determination of around 100 pesticides in dry samples (rice and wheat flour) by ultra-performance LC coupled to a triple quadrupole mass analyzer working in tandem mode (UPLC/QqQ-MS/MS). The sample preparation step was optimized for both matrixes. Pesticides were extracted from rice samples using aqueous ethyl acetate, while aqueous acetonitrile extraction [modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) method] was used for wheat flour matrixes. In both cases the extracts were then cleaned up by dispersive solid phase extraction with MgSO4 and primary secondary amine+C18 sorbents. A further cleanup step with Florisil was necessary to remove fat in wheat flour. The method was validated at two concentration levels (3.6 and 40 µg/kg for most compounds), obtaining recoveries ranging from 70 to 120%, intraday and interday precision values≤20% expressed as RSDs, and expanded uncertainty values≤50%. The LOQ values ranged between 3.6 and 20 µg/kg, although it was set at 3.6 µg/kg for the majority of the pesticides. The method was applied to the analysis of 20 real samples, and no pesticides were detected.

Identification of transformation products of pesticides and veterinary drugs in food and related matrices: use of retrospective analysis.

Retrospective analysis has been applied in different samples, including honey, meat, feed and nutraceutical products from ginkgo biloba, soya, royal jelly and green tea, with the aim of searching transformation products of pesticides and veterinary drugs, which were not included in an initial analysis. Generic extraction and analytical procedures based on high resolution mass spectrometry (Exactive-Orbitrap analyser was used) have been applied. All obtained data have been reprocessed and some compounds as anhydroerythromycin in honey and 3,5,6-trichloro-2-pyridinol in feed have been detected, demonstrating the applicability and the utility of the procedure. Advantages and disadvantages of retrospective approach have been highlighted.

Application of QuEChERS based method for the determination of pesticides in nutraceutical products (Camellia sinensis) by liquid chromatography coupled to triple quadrupole tandem mass spectrometry.

A QuEChERS (quick, easy, cheap, effective, rugged, and safe) based method has been evaluated and validated for the determination and quantification of approximately 100 LC-amenable pesticides in nutraceutical products obtained from green tea (Camellia sinensis). Extraction was performed with acidified acetonitrile (acetic acid 1% (v/v)), and additional clean-up steps were not necessary. Pesticides determination was achieved using ultra high performance liquid chromatography coupled to triple quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). Total running time was 11 min. Pesticides were quantified using matrix-matched calibration. Recoveries ranged from 70% to 117% and relative standard deviation (RSD) was lower than 20% at concentration levels of 25, 50 and 100 µg/kg for intra-day precision and equal or lower than 25% for inter-day precision. Limits of quantification (LOQ) were equal or lower than 25 µg/kg. The validated method was applied to commercial nutraceutical products, detecting acetamiprid (56 µg/kg) and carbendazim (13 µg/kg) in two samples.

Multifamily determination of pesticide residues in soya-based nutraceutical products by GC/MS-MS.

An analytical method based on a modified QuEChERS extraction coupled with gas chromatography-tandem mass spectrometry (GC-MS/MS) was evaluated for the determination of 177 pesticides in soya-based nutraceutical products. The QuEChERS method was optimised and different extraction solvents and clean-up approaches were tested, obtaining the most efficient conditions with a mixture of sorbents (PSA, C18, GBC and Zr-Sep(+)). Recoveries were evaluated at 10, 50 and 100 µg/kg and ranged between 70% and 120%. Precision was expressed as relative standard deviation (RSD), and it was evaluated for more than 160 pesticides as intra and inter-day precision, with values always below 20% and 25%, respectively. Limits of detection (LODs) ranged from 0.1 to 10 µg/kg, whereas limits of quantification (LOQs) from 0.5 to 20 µg/kg. The applicability of the method was proved by analysing soya-based nutraceuticals. Two pesticides were found in these samples, malathion and pyriproxyfen, at 11.1 and 1.5 µg/kg respectively.

Systematic Study of the Content of Phytochemicals in Fresh and Fresh-Cut Vegetables.

Vegetables and fruits have beneficial properties for human health, because of the presence of phytochemicals, but their concentration can fluctuate throughout the year. A systematic study of the phytochemical content in tomato, eggplant, carrot, broccoli and grape (fresh and fresh-cut) has been performed at different seasons, using liquid chromatography coupled to triple quadrupole mass spectrometry. It was observed that phenolic acids (the predominant group in carrot, eggplant and tomato) were found at higher concentrations in fresh carrot than in fresh-cut carrot. However, in the case of eggplant, they were detected at a higher content in fresh-cut than in fresh samples. Regarding tomato, the differences in the content of phenolic acids between fresh and fresh-cut were lower than in other matrices, except in winter sampling, where this family was detected at the highest concentration in fresh tomato. In grape, the flavonols content (predominant group) was higher in fresh grape than in fresh-cut during all samplings. The content of glucosinolates was lower in fresh-cut broccoli than in fresh samples in winter and spring sampling, although this trend changes in summer and autumn. In summary, phytochemical concentration did show significant differences during one-year monitoring, and the families of phytochemicals presented different behaviors depending on the matrix studied.

Determination of quaternary ammonium compounds in oranges and cucumbers using QuEChERS extraction and ultra-performance liquid chromatography/tandem mass spectrometry.

A simple and fast method has been developed for determining relevant quaternary ammonium compounds in cucumber and orange samples. The target compounds were benzoalkonium chloride (BAC-10, BAC-12, BAC-14, and BAC-16), didecyldimethylammonium chloride, and benzethonium chloride, all frequently used biocides in the agrifood industry. An extraction based on the buffered Quick, Easy, Cheap, Effective, Rugged, and Safe method and determination by ultra-performance LC/MS/MS that eluted the biocides in less than 5 min were used. The method was fully validated and implemented in a UNE-EN-ISO/IEC 17025 accredited laboratory for its application to the analysis of real samples. Performance characteristics of the method are reported, including an estimation of measurement uncertainty. Calibration curves were set between 0.01 and 0.150 mg/kg, LOD values were always between 0.4 and 1.0 microg/kg, LOQ values were in the range 1-4 microg/kg, recovery was between 81 and 115%, intraday and interday precision were always lower than 17% (expressed as RSD), and expanded uncertainty was always lower than 40%. The validation was accomplished for the two studied matrixes at spiking concentrations of 0.011 and 0.050 mg/kg. The method has been applied to the analysis of 30 cucumber and orange samples that were found to contain concentrations of BAC-12 that ranged between 0.015 and 0.210 mg/kg and of BAC-14 between 0.018 and 0.081 mg/kg.

QuEChERS approach for the determination of biopesticides in organic and nonorganic vegetables and fruits by ultra-performance liquid chromatography/tandem mass spectrometry.

A method for the determination of 14 biopesticides and piperonyl butoxide (PBO), often applied in organic farming, in vegetables and fruits has been developed. Extraction was performed using the Quick, Easy, Cheap, Effective, Rugged, and Safe method, and the determination was carried out by ultra-performance LC/MS/MS in 9 min. Several different food commodities were evaluated as representative matrixes, namely, cucumber, tomato, pepper (high water content), and strawberry and orange (high acid and water content). Biopesticides were quantified using matrix-matched calibration, and the optimized method was validated at three concentration levels, i.e., 10, 50, and 100 microg/kg, for all the compounds, yielding recoveries in the range 70-112%, 71-112%, and 70-109%, respectively, with RSDs <28%. LOQs were <3 microg/kg for all biopesticides. The validated procedure was applied to the analysis of vegetables and fruits, including products from organic farming; PBO was detected in one sample at 8 microg/kg.