Occurrence of Ergot Alkaloids in Major and Minor Cereals from Northern Italy: A Three Harvesting Years Scenario.

Ergot alkaloids (EAs), mycotoxins produced mainly by fungi of the Claviceps genus, have been frequently reported in rye, while their increasingly frequent occurrence in other cereals is likely related to weather conditions, with the incidence of ergot sclerotia in winter grains being related to heavy rainfall and moist soils at critical periods. However, compared to other regulated mycotoxins, data about the prevalence and occurrence of EAs in major and minor cereals harvested in the Mediterranean growing areas are still scant. In this regard, the current study reported the occurrence of EAs in 18 genotypes of winter cereals harvested over 3 years from an experimental field located in North Italy which were analyzed by HPLC-MS/MS. Results indicate a widespread occurrence of all the major EAs in all the considered cereal crops, especially under supportive meteorological conditions. EA contamination was dependent on the harvest year (p < 0.0001) which was particularly high in 2020 for all the considered species. The results also demonstrated a large co-occurrence of EAs with 98 cereal samples out of 162 contaminated with at least one of the 12 EAs (60% positive samples) in the range LOD: 15,389 µg/kg (median value: 2.32 µg/kg), expressed as the sum of the EAs. Rye was confirmed to be the crop more susceptible to the fungal infection (EAs content up to 4,302 µg/kg). To the best of our knowledge, we have reported the accumulation of EAs in tritordeum (LOD: 15,389 µg/kg) and in emmer (LOD: 1.9 µg/kg) for the first time.

Multi-mycotoxin determination in plant-based meat alternatives and exposure assessment.

The aim of this study was to fill in the gap regarding the occurrence of mycotoxins in plant-based meat alternatives. Hence, a multi-mycotoxin method (aflatoxins, ochratoxin A, fumonisins, zearalenone, and mycotoxins from the Alternaria alternata genera) was developed followed by an exposure assessment for the Italian consumers' exposure to mycotoxins. A total of 13 meat alternatives samples based on soy, pea, chickpea, lupin, and seitan were analysed. With the exception of seitan, all of the remaining samples were contaminated with one mycotoxin or mixtures of up to seven mycotoxins. The level of contamination was as low as 0.2 µg/kg alternariol methyl ether and as high as 66.9 µg/kg fumonisin B1. To analyse the exposure to mycotoxins due to plant-based meat alternatives consumption we used the consumption meat data from the Food and Agriculture Organization for Italian adult consumers and simulated a full replacement of meat with plant-based meat alternatives. Based on our model, consumption of plant-based meat alternatives led to a non-tolerable exposure to alternariol (hazard index (HI) > 1) in pea-based burger and soy + wheat-based steak, while samples contaminated with aflatoxins, respectively ochratoxin A, indicated a health concern related to liver and renal cancer (margin of exposure (MOE) < 10,000). This is the first study that presents the co-occurrence of mycotoxins in multiple plant-based meat alternatives. Moreover, these results indicate that there is a need for policymakers to consider the regulation of mycotoxins in plant-based meat alternatives in order to ensure consumers' safety.

Analytical Methods Based on Liquid Chromatography and Capillary Electrophoresis to Determine Neonicotinoid Residues in Complex Matrices. A Comprehensive Review.

Neonicotinoids (NNIs) are neuro-active and systemic insecticides widely used to protect crops from pest attack. During the last decades, there has been an increase concern about their uses and toxic effects, especially to beneficial and non-target insects such as pollinators. To assess potential health hazards and the environmental impacts derived from NNIs uses, a great variety of analytical procedures for the determination of their residues and their metabolites at trace level in environmental, biological and food samples have been reported. Due to the complexity of the samples, efficient sample pretreatment methods have been developed, which include mostly clean-up and preconcentration steps. On the other hand, among the analytical techniques used for their determination, high-performance liquid chromatography (HPLC) coupled to ultraviolet (UV) or mass spectrometry (MS) detection is the most widely used, although capillary electrophoresis (CE) has also been employed in the last years, considering some improvements in sensitivity when coupling with new MS detectors. In this review, we present a critical overview of analytical methods based on HPLC and CE reported in the last decade, discussing relevant and innovative sample treatments for the analysis of environmental, food and biological samples.

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

Nanofibrous Online Solid-Phase Extraction Coupled with Liquid Chromatography for the Determination of Neonicotinoid Pesticides in River Waters.

Polymeric nano- and microfibers were tested as potential sorbents for the extraction of five neonicotinoids from natural waters. Nanofibrous mats were prepared from polycaprolactone, polyvinylidene fluoride, polystyrene, polyamide 6, polyacrylonitrile, and polyimide, as well as microfibers of polyethylene, a polycaprolactone nano- and microfiber conjugate, and polycaprolactone microfibers combined with polyvinylidene fluoride nanofibers. Polyimide nanofibers were selected as the most suitable sorbent for these analytes and the matrix. A Lab-In-Syringe system enabled automated preconcentration via online SPE of large sample volumes at low pressure with analyte separation by HPLC. Several mat layers were housed in a solvent filter holder integrated into the injection loop of an HPLC system. After loading 2 mL sample on the sorbent, the mobile phase eluted the retained analytes onto the chromatographic column. Extraction efficiencies of 68.8-83.4% were achieved. Large preconcentration factors ranging from 70 to 82 allowed reaching LOD and LOQ values of 0.4 to 1.7 and 1.2 to 5.5 µg·L-1, respectively. Analyte recoveries from spiked river waters ranged from 53.8% to 113.3% at the 5 µg·L-1 level and from 62.8% to 119.8% at the 20 µg·L-1 level. The developed methodology proved suitable for the determination of thiamethoxam, clothianidin, imidacloprid, and thiacloprid, whereas matrix peak overlapping inhibited quantification of acetamiprid.

Sweeping-micellar electrokinetic chromatography with tandem mass spectrometry as an alternative methodology to determine neonicotinoid and boscalid residues in pollen and honeybee samples.

In this work, it is proposed for the first time an electrophoretic approach based on micellar electrokinetic chromatography coupled with tandem mass spectrometry (MEKC-MS/MS) for the simultaneous determination of nine neonicotinoids (NNIs) together with the fungicide boscalid in pollen and honeybee samples. The separation was performed using ammonium perfluorooctanoate (50 mM, pH 9) as both volatile surfactant and electrophoretic buffer compatible with MS detection. A stacking strategy for accomplishing the on-line pre-concentration of the target compounds, known as sweeping, was carried out in order to improve separation efficiency and sensitivity. Furthermore, a scaled-down QuEChERS was developed as sample treatment, involving a lower organic solvent consumption and using Z-Sep+ as dispersive sorbent in the clean-up step. Regarding the detection mode, a triple quadrupole mass spectrometer was operating in positive ion electrospray mode (ESI+) under multiple reaction monitoring (MRM). The main parameters affecting MS/MS detection as well as the composition of the sheath-liquid (ethanol/ultrapure water/formic acid, 50:49.5:0.5 v/v/v) and other electrospray variables were optimized in order to achieve satisfactory sensitivity and repeatability. Procedural calibration curves were established in pollen and honeybee samples with LOQs below 11.6 µg kg-1 and 12.5 µg kg-1, respectively. Precision, expressed as RSD, lower than 15.2% and recoveries higher than 70% were obtained in both samples. Two positive samples of pollen were found, containing imidacloprid and thiamethoxam. Imidacloprid was also found in a sample of honeybees. The obtained results highlight the applicability of the proposed method, being an environmentally friendly, efficient, sensitive and useful alternative for the determination of NNIs and boscalid in pollen and honeybee samples.

A natural deep eutectic solvent as a novel dispersive solvent in dispersive liquid-liquid microextraction based on solidification of floating organic droplet for the determination of pesticide residues.

Current trends in analytical chemistry encourage the use of innocuous solvents to develop modern methods aligned with green chemistry. In this sense, natural deep eutectic solvents (NADESs) have emerged as a novel generation of green solvents which can be employed in sample treatments as an alternative to the toxic organic solvents commonly used so far. In this work, a new extraction method employs dispersive liquid-liquid microextraction based on a solid floating organic droplet (DLLME-SFO), by using a mixture composed of a less dense than water extraction solvent, 1-dodecanol, and a novel dispersive solvent, NADES. The methodology was proposed to extract and preconcentrate some pesticide residues (fipronil, fipronil-sulfide, fipronil-sulfone, and boscalid) from environmental water and white wine samples before analysis by liquid-chromatography coupled to ultraviolet detection (HPLC-UV). Limits of quantification (LOQs) lower than 4.5 µg L-1, recoveries above 80%, and precision, expressed as RSD, below 15% were achieved in both samples showing that the proposed method is a powerful, efficient, and green alternative for the determination of these compounds and, therefore, demonstrating a new application for NADES in sample preparation. In addition, the DLLME-SFOD-HPLC-UV method was evaluated and compared with other reported approaches using the Analytical GREEnness metric approach, which highlighted the greenness of the proposed method.

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry.

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 µg/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 µg/kg).

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 µg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

Capillary liquid chromatography as an effective method for the determination of seven neonicotinoid residues in honey samples.

A new analytical method based on capillary liquid chromatography with diode array detection has been developed for the simultaneous quantification of seven neonicotinoid insecticides commercially available (imidacloprid, thiacloprid, clothianidin, thiamethoxam, acetamiprid, nitenpyram, and dinotefuran) in honey samples. The separation was achieved in a Zorbax XDB-C18 column (150 × 0.5 mm id, 5 µm), with a mobile phase consisting of ultrapure water (solvent A) and acetonitrile (solvent B) at a flow rate of 10 µL/min. Capillary column was thermostated at 25°C during the analysis and 254 or 270 nm was established as detection wavelength, depending on the analyte. Furthermore, full loop injection mode (8 µL) was selected, using water as injection solvent. Finally, the optimized method was applied to the analysis of neonicotinoid residues in honey of different floral origins using dispersive liquid-liquid microextraction as sample treatment. Variables affecting the extraction efficiency were optimized, choosing methanol and dichloromethane as dispersive and extraction solvents, respectively. The method was characterized in terms of linearity ( R 2 ≥ 0.9948), repeatability, reproducibility (relative standard deviation below 4.5 and 6.3% respectively), and recoveries (≥80.5%). Detection and quantification limits were lower than 6.6 and 22.0 µg/kg for the studied analytes, respectively.

Micellar electrokinetic chromatography as efficient alternative for the multiresidue determination of seven neonicotinoids and 6-chloronicotinic acid in environmental samples.

A simple, sensitive, and efficient method has been developed for the determination of the seven neonicotinoid insecticides commercially available (imidacloprid, thiacloprid, clothianidin, thiamethoxam, acetamiprid, nitenpyram, and dinotefuran) and the main metabolite 6-chloronicotinic acid. Micellar electrokinetic chromatography (MEKC) mode was applied, using 48.5 cm of total length capillary (50 µm i.d.) with an extended light-path capillary (150 µm). The running electrolyte consisted of 25 mM sodium tetraborate buffer (pH 9.2) containing 120 mM of sodium dodecyl sulfate and 15% of methanol (v/v). A voltage of 27 kV and a temperature of 25 °C were applied. Samples dissolved in deionized water were hydrodynamically injected at 50 mbar for 12 s, achieving the analysis in less than 12 min. Diode array detection (DAD) was performed at 220, 254, and 270 nm, depending on the analyte. Two different methodologies as sample treatments were developed; for water samples, solid-phase extraction was checked using different cartridges (C18, Oasis® HLB, Oasis® HLB Prime, and Strata-X), being the best option Oasis® HLB for preconcentration and cleanup. In the case of soil samples, a simple solid-liquid extraction was applied using a mixture of 1:3 (v/v) acetonitrile/dichloromethane. Satisfactory linearity, trueness, and precision were achieved, with detection limits in the range of 0.1-0.4 µg L-1 for river water and 1.0-2.9 µg kg-1 for soil samples. Recoveries in the range of 80-107% for all of the assayed neonicotinoids in water samples of different origin and 73-92% for soil samples were achieved.

Screening of extraction properties of nanofibers in a sequential injection analysis system using a 3D printed device.

A novel application of the three-dimensional printing technology for the automation of solid phase extraction procedures in a low-pressure sequential injection analysis system is presented. A 3D printed device was used as a housing for nanofiber membranes in solid phase extraction. The applicability of the device is demonstrated with the extraction of substances of various physical-chemical properties. Pharmaceuticals including non-steroidal anti-inflammatory drugs, antihistaminics, and steroidal structures, as well as emerging pollutants such as bisphenols and pesticide metsulfuron methyl were used as model analytes to study the extraction performance of the nanofibers. Six different nanofiber types comprising polyamide, polyethylene, polyvinylidene fluoride, polycaprolactone combined with polyvinylidene fluoride, and polyacrylonitrile, produced by electrospinning were tested in solid phase extraction. The suitability of specific nanofibers for particular analytes is demonstrated.

A high-throughput UHPLC method for the analysis of 5-nitroimidazole residues in milk based on salting-out assisted liquid-liquid extraction.

A novel salting-out assisted liquid-liquid extraction (SALLE) method has been developed for the extraction of eight 5-NDZ antibiotics from milk samples prior to their analysis by UHPLC. An exhaustive study of the parameters involved in the SALLE procedure has been carried out, optimizing the extraction solvent volume, the amount of the salt agent, and the centrifugation and vortex timing by an experimental design. After sample treatment, the obtained extract was reconstituted in the mobile phase (6:94 (v/v) acetonitrile/water containing 0.1% (v/v) of formic acid) and analyzed by the proposed UHPLC-UV method. Separation was accomplished in a C18 Zorbax Eclipse Plus (50mm×2.1mm, 1.8µm) column at 45°C within 8min. Gradient mode was considered using a mobile phase consisting of 0.1% (v/v) formic acid aqueous solution and acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.45mL/min. Analytical signals were monitored at 320nm. Matrix-matched calibration curves showed satisfactory linearity (R2≥0.996). LODs, ranging from 2 to 4µg/L, were achieved and precision studies showed RSDs lower than 12.8% in terms of peak height. Additionally, recoveries higher than 62.8% were obtained for all studied compounds in milk samples.