Mycotoxins and pyrrolizidine alkaloids in herbal dietary supplements.

The market demand for herbal dietary supplements is rapidly growing and such products are becoming more common and accessible to consumers. However, the knowledge about their safety remains incomplete. Herbal dietary supplements are one of the food groups that can contribute significantly to human health concerns arising from chronic exposure to pyrrolizidine alkaloids and mycotoxins. This study aimed to simultaneously determine 79 natural contaminants, including mycotoxins, as well as pyrrolizidine and tropane alkaloids in herbal dietary supplements in one analytical run. Exposure assessment and human health risks were assessed for all compounds included in this study. The total concentration of naturally occurring contaminants in herbal dietary supplements reached 5.3 mg kg-1 and the most frequently detected mycotoxins were tentoxin and alternariol monomethyl ether. The latter was detected with the highest frequency, reaching concentrations up to 2.5 mg kg-1. The obtained results indicate a potential risk to public health related to herbal dietary supplement consumption.

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.

Determination of Fungi and Multi-Class Mycotoxins in Camelia Sinensis and Herbal Teas and Dietary Exposure Assessment.

In this paper, a study of fungal and multi-mycotoxin contamination in 140 Camellia sinensis and 26 herbal teas marketed in Latvia is discussed. The analysis was performed using two-dimensional liquid chromatography with time-of-flight mass spectrometry (2D-LC-TOF-MS) and MALDI-TOF-MS. In total, 87% of the tea samples tested positive for 32 fungal species belonging to 17 genera, with the total enumeration of moulds ranging between 1.00 × 101 and 9.00 × 104 CFU g-1. Moreover, 42% of the teas (n = 70) were contaminated by 1 to 16 mycotoxins, and 37% of these samples were positive for aflatoxins at concentrations ranging between 0.22 and 41.7 µg kg-1. Deoxynivalenol (DON) and its derivatives co-occurred in 63% of the tea samples, with their summary concentrations reaching 81.1 to 17,360 µg kg-1. Ochratoxin A (OTA), enniatins, and two Alternaria toxins were found in 10-37% of the teas at low concentrations. The dietary exposure assessment based on the assumption of a probable full transfer of determined mycotoxins into infusions indicated that the analysed teas are safe for consumers: the probable maximum daily exposure levels to OTA and the combined DON mycotoxins were only 0.88 to 2.05% and 2.50 to 78.9% of the tolerable daily intake levels.

Pesticide residues in beehive matrices are dependent on collection time and matrix type but independent of proportion of foraged oilseed rape and agricultural land in foraging territory.

Pesticide residues in bee products is still a major issue. However, the relations to botanical source and land use characteristics are not clear. The large variability of residues detected questions the suitability of bee-collected- and other hive materials as indicators for environmental contamination. The aim of our study was to clarify whether different beehive matrices contain similar pesticide residues, and how these are correlated with forage preferences and land use types in foraging areas. We tested bee-collected pollen, beebread, honey, nurse bees and honey bee larvae for the presence of concurrently used agricultural pesticides in Estonia. Samples were collected at the end of May and mid-July to include the main crop in northern region - winter and spring oilseed rape (Brassica napus). We saw that different beehive matrices contained various types of pesticide residues in different proportions: pollen and beebread tended to contain more insecticides and fungicides, whereas herbicides represented the primary contaminant in honey. The variations were related to collection year and time but were not related to crops as basic forage resource nor the land use type. We found few positive correlations between amount of pesticides and proportion of pollen from any particular plant family. None of these correlations were related to any land-use type. We conclude that pesticide residues in different honey bee colony components vary largely in amount and composition. The occurrence rate of pesticide residues was not linked to any particular crop.

Simultaneous screening and quantification of aminoglycoside antibiotics in honey using mixed-mode liquid chromatography with quadrupole time-of-flight mass spectroscopy with heated electrospray ionization.

An analytical method based on liquid chromatography with quadrupole time-of-flight mass spectrometry has been developed for the simultaneous determination of six aminoglycoside antibiotics in honey. The sample pretreatment included extraction with aqueous trichloroacetic acid followed by solid-phase extraction on Strata-X polymeric reversed phase cartridges. Liquid chromatography separation was performed on an Obelisc R zwitterionic type mixed-mode column. An ionBooster™ heated electrospray source was used and showed enhanced ionization efficiency in comparison to a conventional electrospray source. The observed signal enhancement ranged from 3- (neomycin) to 16-fold (gentamicin C1). A data-dependent mass spectrometry acquisition approach was employed, in which the full mass spectrometry dataset provided quantification and a scheduled precursor list was used to trigger an alternating data-dependent acquisition of MS2 spectra for confirmation purposes. The described method was validated in accordance to CD 2002/657/EC. Decision limit values were in the range 11.2-33.6 ng/g, and satisfactory performance characteristics were obtained for recovery (65-76%), repeatability (3.8-7.3%), and linearity (≥0.995). The method was applied to the analysis of 49 real honey samples from the country of Georgia. Streptomycin was detected in two samples at 117 and 35 ng/g, and gentamicin C1 was detected in one sample at 32 ng/g.

The effects of ultrasonication, fermentation with Lactobacillus sp., and dehydration on the chemical composition and microbial contamination of bovine colostrum.

The aim of this study was to evaluate the influence of ultrasonication, fermentation with Lactobacillus plantarum LUHS135 and Lactobacillus paracasei LUHS244, and different methods of dehydration on the chemical composition of bovine colostrum (BC), including the fatty acid and free amino acid profile and the content of micro- and macroelements. In addition, we analyzed the changes in lactic acid bacteria count, microbial contamination (aerobic mesophilic spore-forming bacteria, enterobacteria including Escherichia coli, and fungi/yeasts), the abundance of biogenic amines, and the concentration of nucleotide monophosphates. Significant effects of different treatments on the free amino acid profile were established, and an increase of lysine concentration by 1.2 to 95.9% was observed in treated BC. All of the treatments reduced the concentration of cadaverine, histamine, and tyramine in BC. The concentrations of macro- and microelements in BC followed the following order Ca > Na > K > Mg and Zn > Fe > Sr > Ba > Mn > Cu > Al > Se > Mo > Cr > Ni > Sn > Co > Pb > Cd. By combining the fermentation with Lactobacillus plantarum strain LUHS135 and vacuum drying, it was possible to increase the abundance of nucleotide monophosphates by more than 100%. All of the treatments reduced the microbial contamination of BC. Thus, the combination of ultrasonication, fermentation, and dehydration can be used for improving the properties and safety of BC.

Improved sensitivity of ochratoxin A analysis in coffee using high-performance liquid chromatography with hybrid triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS).

A novel and sensitive method utilising high-performance liquid chromatography coupled to triple quadrupole-linear ion trap mass spectrometry (LC-QqQLIT-MS/MS) was developed in order to analyse the content of ochratoxin A (OTA) in coffee samples. The introduction of the triple-stage MS scanning mode (MS(3)) has been shown to increase greatly sensitivity and selectivity by eliminating the high chromatographic baseline caused by interference of complex coffee matrices. The analysis included the sample preparation procedure involving extraction of OTA using a methanol-water mixture and clean-up by immunoaffinity columns and detection using the MS(3) scanning mode of LC-QqQLIT-MS/MS. The proposed method offered a good linear correlation (r(2) > 0.998), excellent precision (RSD < 2.9%) and recovery (94%). The limit of quantification (LOQ) for coffee beans and espresso beverages was 0.010 and 0.003 µg kg(-1), respectively. The developed procedure was compared with traditional methods employing liquid chromatography coupled to fluorescent and tandem quadrupole detectors in conjunction with QuEChERS and solid-phase extraction. The proposed method was successfully applied to the determination of OTA in 15 samples of coffee beans and in 15 samples of espresso coffee beverages obtained from the Latvian market. OTA was found in 10 samples of coffee beans and in two samples of espresso in the ranges of 0.018-1.80 µg kg(-1) and 0.020-0.440 µg l(-1), respectively. No samples exceeded the maximum permitted level of OTA in the European Union (5.0 µg kg(-1)).

Development of a sensitive method for the determination of acrylamide in coffee using high-performance liquid chromatography coupled to a hybrid quadrupole Orbitrap mass spectrometer.

The emerging trend towards high-resolution mass spectrometry (MS) alternatives was evaluated by the application of Orbitrap MS for the determination of acrylamide in coffee samples. The high resolving power of the Orbitrap MS provided the high selectivity and sensitivity that enabled quantitative analysis of acrylamide in complex matrices, such as coffee. Several sample preparation methods and scanning modes of the MS (full MS, t-SIM, t-MS2) were assessed in order to optimise parameters of the analytical method. The final procedure involved the extraction of acrylamide with acetonitrile, solid-phase extraction with dispersive primary secondary amine (PSA) and amino columns, and the detection by ultra-performance liquid chromatography coupled to a hybrid quadrupole-Orbitrap MS (HPLC-Q-Orbitrap) operated in targeted MS2 scanning mode. The repeatability of the method at the lowest calibration level (10 µg kg(-1)), expressed as relative standard deviation, was 7.8% and the average recovery of acrylamide was 111%. The proposed method was applied to the determination of acrylamide in 22 samples of roasted coffee obtained from the Latvian retail market. Acrylamide concentration in coffee samples was in the range of 166-503 µg kg(-1).

The role of nutrients in the biodegradation of 2,4,6-trinitrotoluene in liquid and soil.

The widely used explosive 2,4,6-trinitrotoluene (TNT) has residues that are potentially explosive, toxic, and mutagenic. TNT and other explosives can be degraded by microorganisms; however, biostimulation is needed for process efficiency. To investigate the effectiveness of using biostimulation to degrade TNT, we added varying concentrations of a nutrient amendment consisting of inorganic salts, plant extracts, and molasses to soil and liquid media. For the inoculum we used a consortium of bacteria AM 06 that had exhibited the ability to degrade TNT and which had been previously isolated from explosives-contaminated soils. Phylogenetically, the clones clustered into seven different genera: Klebsiella, Raoultella, Serratia, Stenotrophomonas, Pseudoxanthomonas, Achromobacter and Pseudomonas. The addition of AM 06 consortium to a liquid environment along with 100% nutrient amendment decreased the amount of TNT (and its degradation products) by up to 90% after 14 days incubation. At the total amount of TNT was less than 100 mg/l, the concentration of TNT did not influence the amount of sugar consumed by the bacteria consortium. In soil media, the TNT degradation process was dependent on the concentration of nutrient amendment added. At higher initial concentrations of TNT (500 mg/kg), bioaugmentation (i.e., addition of bacteria inoculum) had a demonstrated effect, especially when nutrient concentrations of 50% and 100% were added to the soil. Findings of this study could further the understanding of the TNT biodegradation processes in water and soil and provide for optimization of the technological conditions for bioremediation.