Metabolic Response of the Yeast Candida utilis During Enrichment in Selenium.

Selenium (Se) was found to inhibit the growth of the yeast Candida utilis ATCC 9950. Cells cultured in 30 mg selenite/L supplemented medium could bind 1368 µg Se/g of dry weight in their structures. Increased accumulation of trehalose and glycogen was observed, which indicated cell response to stress conditions. The activity of antioxidative enzymes (glutathione peroxidase, glutathione reductase, thioredoxin reductase, and glutathione S-transferase) was significantly higher than that of the control without Se addition. Most Se was bound to water-insoluble protein fraction; in addition, the yeast produced 20-30 nm Se nanoparticles (SeNPs). Part of Se was metabolized to selenomethionine (10%) and selenocysteine (20%). The HPLC-ESI-Orbitrap MS analysis showed the presence of five Se compounds combined with glutathione in the yeast. The obtained results form the basis for further research on the mechanisms of Se metabolism in yeast cells.

Quantitative determination of pesticide residues in specific parts of bee specimens by nanoflow liquid chromatography high resolution mass spectrometry.

The presence of pesticide residues in bees is of great interest, given the central role of bees as indicators for environmental assessment. The goal of this article is to propose a method to capture enhanced chemical information for these central environmental indicators. Most of the methods rely on the analysis of pooled samples rather than individual specimens due to practical sample preparation method considerations and limitations in sensitivity. This leads to miss information on the mapping of pesticides and actual amount of pesticide per specimen. In this article, a nanoflow liquid chromatography system coupled to high resolution mass spectrometry (using a hybrid quadrupole-Orbitrap instrument) has been applied for the development of a multiresidue pesticide method for the determination of 162 multiclass pesticides in specific part of honeybee samples (ca. abdomen, head or thorax). The reduced flow rate provided an enhancement in sensitivity and a strong reduction of matrix effects, thus only a quick and simple ultrasound assisted extraction using minute amount of sample was required. Satisfactory results were obtained for all tested analytes with concentration levels detected lower than 0.5 ng g-1 in all cases, thus being acceptable for monitoring purposes. Matrix effect was negligible for 94% of compounds. Extraction recoveries ranged from 70% to 105%, being within SANTE guidelines. Finally, the applicability of the method was demonstrated, by successful application to the analysis of contaminated honeybee samples, extracting useful information from specific bee parts of single specimens, thus, enabling pseudo spatially resolved chemical information.

Basin-scale monitoring and risk assessment of emerging contaminants in South American Atlantic coastal lagoons.

Emerging contaminants (ECs) such as pharmaceuticals, personal care products, drugs of abuse and polar pesticides are under particular attention due to their high consumption, frequent detection in the environment and reported ecotoxicological risk. This study investigates the occurrence and distribution of multiclass of ECs in surface waters at basin scale of two Atlantic coastal lagoons of Uruguay, South America. For this purpose, a target screening approach covering up to 362 compounds was employed using nanoflow liquid chromatography - high resolution mass spectrometry (nanoLC/HRMS). 56 compounds were identified including five banned pesticides in the European Union: atrazine, carbendazim, chlorpyrifos ethyl, diazinon, and ethion. Pharmaceuticals, hormones and drugs of abuse showed maximum detection frequencies and concentrations downstream cities. The highest occurrence of pesticides was found in lagoons and streams with neighboring agricultural activity. ECs were also found in coastal sea. Environmental risk assessment revealed that the hormones 17α-ethinylestradiol and 17-ß-estradiol showed the highest risk to aquatic organisms in these basins. This study represents the first basin- scale monitoring of ECs in superficial waters encompassing streams, lagoons, and coastal seas in Uruguay, South America.

Use of a modified QuEChERS method for the determination of mycotoxin residues in edible nuts by nano flow liquid chromatography high resolution mass spectrometry.

A nanoflow liquid chromatography high resolution mass spectrometry method for the quantification of mycotoxins in nuts has been developed. Two strategies based on QuEChERS methodology were evaluated. Thus, EMR-lipid was compared with a conventional mixture of PSA and C18 dispersive solid phase extraction sorbents which have been commonly used in this type of matrices as sample clean-up. The results showed that the use of EMR-lipid reduced more effectively matrix components, achieving a negligible matrix effect for all mycotoxins studied in peanut, pistachio and almond. The proposed method was validated in line with SANTE guidelines using EMR-Lipid as dispersive solid phase extraction sorbent. The lowest concentration level were between 0.05 and 5 µg kg-1, being lower than the maximum levels established by the current legislation. Recovery rates ranged from 75% to 98% was obtained in all sample studied, achieving also satisfactory precision with RSD values lower than 19% in all cases.

Multi-residue pesticide analysis in virgin olive oil by nanoflow liquid chromatography high resolution mass spectrometry.

In this article, a nanoflow liquid chromatography system coupled to high resolution mass spectrometry (nanoflow LC/ESI Q-Orbitrap-MS) has been applied for the development of a multiresidue pesticide method for the determination of 162 multiclass pesticides in olive oil samples. Due to the relatively high lipid content of the raw QuEChERS acetonitrile extracts obtained from this type of fatty vegetable samples, a dispersive solid phase extraction (dSPE) sorbent proposed to retain both fatty acids and triglycerides, namely Enhanced Matrix Removal-Lipid (EMR-Lipid) has been implemented as additional cleanup step. The analytical performances of the proposed method were evaluated,achieving recoveries in the range 75-119% with relative standard deviations lower than 19% (n = 6). The dSPE sorbent allowed the removal of most coextracted interferences without a significant loss of analytes. Matrix effects were also evaluated, showing a negligible effect for most of the compounds tested, when a dilution factor of 50 was applied. Notably, despite the use of relatively high dilution factors (e.g. 1:50) to minimize matrix effects, the lowest concentration levels detected with this method - in the low µg kg-1 range - are well below the corresponding maximum residue levels established by the current European legislation.

Dilute-and-shoot coupled to nanoflow liquid chromatography high resolution mass spectrometry for the determination of drugs of abuse and sport drugs in human urine.

In this work, a sensitive nanoflow liquid chromatography high-resolution mass spectrometry screening method has been developed for the determination of multiclass drugs of abuse and sport drugs in human urine. 81 drugs belonging to different multiclass pharmaceuticals were targeted. The method is based on the use of a nanoLC column (75 µm × 150 mm, 3 µm particle size and 100 Špore) with the nanospray emitter tip integrated so that dead volumes are significantly minimized. Data acquisition method included both full-scan and all ion fragmentation experiments using an Orbitrap analyser (Q-Exactive) operated in the positive ionization mode. To increase laboratory throughput, a dilute-and-shoot methodology has been tested and proposed, based solely on direct urine dilution without further sample workup. Matrix effects were evaluated, showing a negligible effect for all studied compounds when a dilution 1:50 was implemented. Despite this high-dilution factor, limits of quantification were still satisfactory, with values below 5 µg L-1 in most cases, being lower than their minimum required performance limits correspond established by the World Anti-Doping Agency. Therefore, the use of the dilute-and-shoot method with the enhanced sensitivity provided by nanoflow LC setup could be useful tool for the determination of studied compounds in drug testing, thus increasing laboratory performance, because a minimum sample treatment steps are required.

Matrix-effect free multi-residue analysis of veterinary drugs in food samples of animal origin by nanoflow liquid chromatography high resolution mass spectrometry.

In this work, a sensitive method based on nanoflow liquid chromatography high-resolution mass spectrometry has been developed for the multiresidue determination of veterinary drugs residues in honey, veal muscle, egg and milk. Salting-out supported liquid extraction was employed as sample treatment for milk, veal muscle and egg, while a modified QuEChERS procedure was used in honey. The enhancement of sensitivity provided by the nanoflow LC system also allowed the implementation of high dilution factors as high as 100:1. For all matrices tested, matrix effects were negligible starting from a dilution factor of 100, enabling, thus, the use of external standard calibration instead of matrix-matched calibration of each sample, and the subsequent increase of laboratory throughput. At spiked levels as low as 0.1 or 1 µg kg-1 before the 1:100 dilution, the obtained signals were still significantly higher than the instrumental limit of quantitation (S/N 10).

Sensitive Detection of Neonicotinoid Insecticides and Other Selected Pesticides in Pollen and Nectar Using Nanoflow Liquid Chromatography Orbitrap Tandem Mass Spectrometry.

In this work, a new method based on nanoflow LC with high-resolution MS was developed for the determination of eight pesticides in pollen and nectar samples, including neonicotinoid insecticides and other selected pesticides commonly found in bees and beeswax. Detection was undertaken with a hybrid quadrupole-Orbitrap mass spectrometer (Q Exactive™) equipped with a commercial nanospray ion source. The extraction of pesticides from pollen samples was performed by a modified micro-QuEChERS method scaled down to Eppendorf tubes, whereas nectar samples were simply diluted with a water-methanol (95 + 5, v/v) solution. Good linearity (>0.999 in all cases) was obtained between 0.05 and 500 µg/kg and between 0.04 and 400 µg/kg for pollen and nectar, respectively. Recovery rates in pollen ranged from 85 to 97%, with RSDs <12%. Matrix effect was evaluated and showed negligible effects for all studied pesticides. The lowest concentration levels tested and validated were 0.5 and 0.4 µg/kg for pollen and nectar matrixes, respectively. In addition, selected incurred samples were studied, obtaining several positive findings in pollen and nectar samples, demonstrating the sensitivity and applicability of the proposed method.

Matrix-effect free quantitative liquid chromatography mass spectrometry analysis in complex matrices using nanoflow LC with integrated emitter tip and high dilution factors.

Matrix effects are probably the Achilles heel of most quantitative liquid chromatography mass spectrometry (LC-MS) methods based on electrospray ionization. This work reports the evaluation of matrix effects in challenging matrices such as food extracts, human urine or wastewater at different dilution factors using nanoflow liquid chromatography-high resolution mass spectrometry (LC-MS). For this purpose, a suite of representative low-molecular weight compounds such as pesticides, drugs of abuse, sport drugs or environmental contaminants were selected. The approach is based on the use of reversed-phase C18 nano columns furnished with an integrated emitter tip. The nanoflow LC system was combined with full-scan high resolution mass spectrometry using a HRMS (orbitrap) instrument operated at a resolution of 70000. The sensitivity achieved with this configuration enables the implementation of high dilution factors (e.g. 1:20, 1:50 or beyond). When combining nanoflow LC-MS analysis with such high dilution factors (e.g. 1:50), signal suppression was negligible in most cases, so that matrix-matched standards may eventually be skipped, simplifying laboratory workflows by using external calibration in demanding applications such as drug analysis in urine, environmental contaminants in wastewater or pesticide testing in food, thus, eliminating the need for standard addition, matrix-matched calibration or isotopically-labelled standards.