Application of Baltic Pine (Pinus sylvestris) Needle Extract as a Gut Microbiota-Modulating Feed Supplement for Domestic Chickens (Gallus gallus).

The valorization of wood industry residues is very desirable from a circular economy perspective. Pine needle extracts are known for their health-promoting properties and therefore can be used as herbal remedies and nutritional supplements. Since the withdrawal of antibiotics as growth promoters in the European Union, natural feed additives that improve poultry health and production are needed. It was proposed that pine needle extract could be a good alternative to antibiotic usage at sub-therapeutic concentrations. The results relevant to our assumption could be obtained by using domestic chickens as an in vivo model for the evaluation of gut microbiota-altering properties of pine needle extract as an herbal supplement. We tested the antimicrobial effects of Baltic pine (Pinus sylvestris) needle extract. Then, we used chicken (Gallus gallus) that received feed supplemented with two different concentrations of the extract for 40 days to evaluate the changes in gut microbiota using 16S rRNA gene sequencing. This preliminary study demonstrated trends toward dose-dependent desirable changes in broiler microbiome, such as a reduction in the relative abundance of Campylobacter.

Nanoflow liquid chromatography mass spectrometry method for quantitative analysis and target ion screening of pyrrolizidine alkaloids in honey, tea, herbal tinctures, and milk.

A method for the determination of pyrrolizidine alkaloids in tea, honey, herbal tinctures, and milk samples was developed by employing nano-LC-MS with high-resolution Orbitrap mass spectrometry. Quantitation was performed using the available analytical standards, and a MS2 target ion screening approach was developed using fragment ions that were specific for pyrrolizidine alkaloids under collision-induced dissociation. Proof of concept was delivered for the screening approach, proposing that the C6H8N+ fragment ion is a highly selective fragment ion for the detection of potential pyrrolizidine alkaloids. The elaborated quantitation was applied for the occurrence study of pyrrolizidine alkaloids in food products available on the Latvian market, including samples of tea (n = 15), honey (n = 40), herbal tinctures (n = 15), and milk (n = 10). The median LOQ over all analytes was 0.33 µg kg-1 in honey, 3.6 µg kg-1 in tea, 3.3 µg kg-1 in herbal tinctures, and 0.32 µg kg-1 in milk. The herbal tinctures samples and milk samples did not contain pyrrolizidine alkaloids above LOQ values. Analytes were detected in 33% of honey and 47% of tea samples. Most common were echimidine, intermedine, and enchinatine N-oxide. Pyrrolizidine alkaloids in tea samples were mainly N-oxides, with the highest total concentration being 215 µg kg-1 among the samples, exceeding the maximum limit of 200 µg kg-1 set by Commission Regulation (EU) 2020/2040. In honey samples, lycopsamine-type alkaloids were detected most frequently, with the highest total concentration equal to 74 µg kg-1. Advantages of the developed nano-LC-MS methods included increased sensitivity in comparison with conventional flow LC-MS, low solvent consumption typical with nano-LC and the novel use of a selective common target ion for detection and discovery of potential pyrrolizidine alkaloids using high resolution mass spectrometry.

Application of Wastewater-Based Epidemiology for Tracking Human Exposure to Deoxynivalenol and Enniatins.

Wastewater-based epidemiology (WBE) is a promising biomonitoring approach with the potential to provide direct information on human intake and exposure to food contaminants and environmental chemicals. The aim of this study was to apply WBE while employing the normalization method for exploring human exposure to selected mycotoxins according to population biomarker 5-hydroxyindoleacetic acid (5-HIAA). This type of normalization technique has been previously used to detect various other compounds. However, to the best of our knowledge, this is the first study tracking human exposure to mycotoxins. A sensitive analytical methodology was developed to achieve reliable quantification of deoxynivalenol, enniatins, and beauvericin in wastewater (WW) samples. The applicability of the method was evaluated by testing 29 WW samples collected at WW treatment plants in Latvia. With frequency of detection greater than 86%, enniatins B, B1, A, and A1 were revealed in WW samples. The estimated total daily intake for enniatins was in the range of 1.8-27.6 µg/day per person. Free deoxynivalenol (DON) was determined in all analysed WW samples. Based on the average 5-HIAA excretion level and the determined 5-HIAA content in the samples, the intake of DON by the human population of Riga was estimated at 325 ng/kg b.w. day.

Two-dimensional liquid chromatography - mass spectrometry as an effective tool for assessing a wide range of pharmaceuticals and biomarkers in wastewater-based epidemiology studies.

The biomarkers used in wastewater-based epidemiology studies have a wide range of physicochemical properties, making simultaneous analysis extremely challenging or even impossible. So far, the majority of analytical procedures employed in this field have focused on specific classes or a limited number of analytes. The heart-cutting two-dimensional liquid chromatography tandem mass spectrometry technique (2D-LC-MS/MS) has enabled the incorporation of compounds with significantly different polarities in a single analytical method. In this study, a 2D-LC-MS/MS method has been developed and optimized for the simultaneous analysis of various biomarkers in wastewater, providing relevant information about lifestyle habits (the usage of alcohol, tobacco, and caffeine), health and well-being (pharmaceuticals), and a population size biomarker (5-HIAA) that is useful for correct calculation of human population during wastewater-based epidemiology studies. A single-laboratory validation procedure showed satisfactory analytical performance, with the mean trueness for most compounds falling in the range of 80÷110 % and the mean repeatability being less than 20 %. The applicability of the method was evaluated by testing 28 wastewater samples collected at different wastewater treatment plants in Latvia. The obtained results revealed the occurrence of 44 out of 62 selected biomarkers, with the lowest quantified concentration at 1.1 ng L-1 in the case of xylometazoline and up to 148 µg L-1 in the case of metformin and 156 µg L-1 for caffeine.

LC-MS/MS characterisation and determination of dansyl chloride derivatised glyphosate, aminomethylphosphonic acid (AMPA), and glufosinate in foods of plant and animal origin.

Glyphosate and other polar and acidic pesticides have been particularly studied due to the concerns over widespread and intensive use. The chemical properties of these compounds necessitate use of customised methods, such as derivatisation or ion exchange chromatography. These approaches present a compatibility problem with ESI-MS due to presence of salts and non-volatile compounds. For that reason, a simple procedure has been developed for the extraction, pre-column derivatisation with dansyl chloride (5-(dimethylamino)naphthalene-1-sulfonyl chloride), and mass spectrometric detection of glyphosate, AMPA, and glufosinate after the separation on a C18stationary phase. The dansyl derivatives were characterised with ESI-MS and their separation from derivatisation reagent byproducts was demonstrated with UV absorption detection. Reagent byproducts eluted before the analytes and were separated from the analytes completely, thus the proposed procedure did not contaminate the mass spectrometers. The proposed procedure was evaluated with respect to the matrix effects and extraction efficiency, and was validated with different mass spectrometers for milk, cucumber, honey, porridge formula, bovine kidney and liver matrix. The LOQ was 10 µg kg-1 for AMPA and glufosinate, and 10-25 µg kg-1for glyphosate, depending on matrix. Measurement uncertainties ranged from 4 to 44%. Method performance was compared to the QuPPe (Quick Polar Pesticides) procedure in combination with a diethylamino-based column from Waters™. In the case of Orbitrap™ detection, the proposed procedure had a comparable performance to the QuPPe procedure. Although, improved peak shape, higher absolute peak intensity, and lower standard deviation of the calibration curve slope was observed with the proposed procedure. This could be explained by the superior electrospray stability and lower extent of ion suppression.

Mycotoxins in cereals and pulses harvested in Latvia by nanoLC-Orbitrap MS.

Twenty-seven mycotoxins in unprocessed cereals (n = 110) and pulses (n = 23) harvested in Latvia were analysed by nanoflow liquid chromatography combined with Orbitrap high-resolution mass spectrometry. One or more mycotoxins were found in 99% of the cereals and 78% of the pulses. Deoxynivalenol, zearalenone and T-2 and HT-2 toxins were prevalent in 9 to 86% of the cereals, mostly below their maximum levels as set by the European regulations. Non-regulated type A and B trichothecenes were prevalent in 5 to 87% of the cereals, at concentrations of 0.27-83 µg kg-1 and 1.7-4,781 µg kg-1, respectively. Quantification of emerging mycotoxins was also provided. Enniatins were detected in 94% of the cereals (3.5-2,073 µg kg-1) and 13% of the pulses (4.4-17 µg kg-1). Alternaria toxins were prevalent in 94% of the cereals at concentrations of 0.72-307 µg kg-1 and in 39% of the pulses at 0.69-10 µg kg-1.

The Occurrence and Dietary Exposure Assessment of Mycotoxins, Biogenic Amines, and Heavy Metals in Mould-Ripened Blue Cheeses.

The occurrence and dietary exposure assessment of 16 mycotoxins, 6 biogenic amines (BAs), and 13 metallic elements in blue-veined cheeses (n = 46) is reported. Co-occurrence of mycophenolic acid (≤599 µg·kg-1) with roquefortine C (≤5454 µg·kg-1) was observed in 63% of the tested cheeses, while BAs were frequently present at concentrations between 0.2 and 717 mg kg-1. The concentrations of heavy metals in cheeses were very low. Chronic/acute exposure assessment based on consumption data from different European populations indicated that the levels of mycotoxins and heavy metals are safe to consumers, whereas, rather high hazard indexes (HI up to 0.77) were determined for BAs according to the worst-case scenario based on high consumption and 95th percentile occurrence. A more detailed acute dietary intake study indicated that histamine and tyramine were predominant among these BAs, reaching 27 and 41% of the acute oral intake reference doses.

Modeling the mobility of glyphosate from two contrasting agricultural soils in laboratory column experiments.

Glyphosate (GLP) currently is one of the most widely used herbicides worldwide. The persistence of GLP and its major metabolite, aminomethylphosphonic acid (AMPA) in the environment has been described by other authors. This study was aimed at comparing the GLP and AMPA behavior in sandy and loamy sand soils after spiking with enhanced (445 µg g-1) concentrations of GLP in herbicide KLINIK® (Nufarm, Austria) and bioaugmentation followed by 40 days weathering and a consistent three-stage leaching in a laboratory column experiment. Soil samples were obtained from mineral topsoil (0-10 cm) within former agricultural lands where soil parent material was formed by glacigenic deposits. The total amount of GLP and AMPA collected during three leaching stages was significantly (p<.05) higher from columns with sandy soil, compared to loamy sand soil. Bioaugmentation resulted in considerably lower concentrations of AMPA in leachates, especially in the sets with sandy soil (p=.01). Leachates were tested using FTIR spectroscopy and Daphnia magna. Statistical analysis of the changes in Ntot, Ctot, K+, Mg2+, Al3+, Ca2+, Mn2+ and Fe3+ concentrations in soils after the leaching experiment revealed that the loamy sand soil was likely to be more sensitive to the addition of GLP and bioaugmentation than sandy soil.

Evaluation of selected buffers for simultaneous determination of ionic and acidic pesticides including glyphosate using anion exchange chromatography with mass spectrometric detection.

Ion chromatography coupled with mass spectrometry is an established technique for determination of ionic analytes, however, sophisticated buffer removal equipment is required to eliminate inorganic compounds from the eluate before introduction into the ion source of mass spectrometer. A standard high-performance liquid chromatography coupled with tandem mass spectrometry setup using an ion exchange column (Metrosep® A Supp 5) is proposed as an alternative approach. For that reason, some buffers including non-volatile carboxylic acid based solutions have been evaluated for simultaneous trace determination of ionic and acidic pesticides including glyphosate in the same extract without a need for sophisticated buffer removal equipment. Two differently designed ionisation sources were compared qualitatively for the application of non-volatile buffers. The study revealed that the choice of buffers had a strong influence on matrix effects in case of spiked extract injections. Finally, pesticides with very different physicochemical properties (logP < 0, logP ≥ 0) and structures (containing carboxylate, phosphonate, azolide, azanide, phenolate, bromate, and chlorate moieties) were quantified in spiked beer and oat extracts with acceptable recoveries (80-110%) using tandem mass spectrometry detection with AB SCIEX QTRAP 5500 instrument after separation using edetate buffer.

Recent Applications of Carbonaceous Nanosorbents in Solid Phase Extraction for the Determination of Pesticides in Food Samples.

This article covers recent trends of carbonaceous nanomaterial supported solid phase extraction for the liquid and gas chromatography analysis of pesticides in food samples. Several advanced carbonaceous nanosorbents have been reported over the previous decade for pesticide extraction, such as graphene derivatives modified by combination with silica, amines, polymers, and/or magnetic nanoparticles. The advances in this field have opened possibilities for using neat or modified carbon nanotubes or graphene-type sorbents for the pre-concentration of polar or nonpolar pesticides by solid phase extraction (SPE) or micro-SPE methods or to apply such nanomaterials for the removal of matrix components by dispersive SPE during sample clean-up. The applications of such nanosorbents should increase in the next few years, considering the expected improvements of their properties, including time and resource economy, selectivity, and reusability.

Development and optimization of confirmatory liquid chromatography-Orbitrap mass spectrometry method for the determination of 17 anticoccidials in poultry and eggs.

A new sensitive and selective multi-residue method based on liquid chromatography - Orbitrap high resolution mass spectrometry (LC-Orbitrap-HRMS) was developed and validated for the determination of 17 anticoccidials in poultry and eggs. Instrumental parameters were optimized by the means of statistical experimental designs to improve the sensitivity, precision, and repeatability of the method. Further optimization of auto-tuned MS parameters led to an increase of signal intensity by 10% to 99% for 16 out of 17 analytes. The sample preparation procedure included extraction from muscle tissue and egg samples with acetonitrile, followed by preconcentration, reconstitution, and filtration. Validation was performed according to the Commission Decision 2002/657/EC. The occurrence of anticoccidials in eggs and poultry was assessed by using the developed analytical procedure within the Latvian national monitoring program, revealing quantifiable residues for 6 analytes (marker residue of nicarbazin - 4,4'-dinitrocarbanilide (DNC), salinomycin, narasin, toltrazuril, and its two metabolites).

Occurrence of glyphosate in beer from the Latvian market.

A sensitive LC-MS/MS method for the determination of glyphosate in beer has been developed, validated, and applied to analyse 100 beer samples from 24 different producers and distributors in Latvia. The selected samples represented most beer brands and varieties sold in local supermarkets. Different procedures for sample preparation and chromatographic separation were compared. The final version of the method consisted of solid phase extraction, chromatographic separation on aminopropyl stationary phase, and detection using tandem mass spectrometry. The concentration of glyphosate in beer varied from below the LOD of 0.2 µg kg-1 up to 150 µg kg-1, higher than previously reported. Significantly higher (p < 0.01) content of glyphosate was observed in beers that did not have the country of production disclosed on the label and were sold in local supermarkets by distributors from Latvia (1.8 µg kg-1 median concentration in locally produced beer, 6.7 µg kg-1 in beer of undisclosed origin).