Caffeine Decreases Topiramate Levels in Zebrafish Larvae in a Pentylenetetrazol-Induced Seizure Model.

Epilepsy ranks as the second-most prevalent neurological disease, and is characterized by seizures resulting in neurobiological and behavioral impairment. Naturally occurring in coffee beans or tea leaves, the alkaloid caffeine (CAF) is the most prevalent global stimulant. Caffeine has been observed to influence epileptic seizures and the efficacy of antiepileptic medications, with a notable impact on topiramate (TPM). This study aimed to explore the influence of CAF on TPM's anticonvulsant effects in zebrafish larvae within a PTZ-induced seizure model, concurrently determining TPM concentrations through a sophisticated analytical approach based on ultrahigh-performance liquid chromatography and subsequent mass spectrometric detection. Zebrafish larvae four days post-fertilization were incubated for 18 h with varying doses of TPM or combinations of CAF + TPM, and locomotor activity was then assessed. Seizures were induced by introducing a PTZ solution to achieve a final concentration of 20 mM. Utilizing liquid chromatography-mass spectrometry (LC-MS/MS), TPM levels in the larvae were quantified. CAF co-administration (especially in higher doses) with TPM caused a decrease in the average locomotor activity in the larvae compared to TPM alone. Moreover, CAF decreased TPM levels in the larvae at all investigated doses. In conclusion, these findings offer a novel perspective on the interplay between CAF and TPM, shedding light on previously unexplored facets. The potential impact of CAF consumption in assisting with epileptic seizure control, unless proven otherwise, suggests a noteworthy consideration for future research and clinical practices.

Absolute quantification of targeted rabbit liver- and meat tissue-specific peptide markers in highly processed food products.

A highly sensitive and selective method for the simultaneous absolute quantification of peptides unique to rabbit meat- and liver-specific tissue was developed using liquid chromatography - triple quadrupole mass spectrometry. Two rabbit skeletal muscle-specific peptides (SSVFVADPK and PHSHPALTPEQK), three rabbit liver tissue-specific peptides (FNLEALVTHTLPFEK, AILNYVANK, and TELAEPTSTR) and one peptide specific to both rabbit offal and skeletal muscle tissue (AFFGHYLYEVAR) were monitored. Analyses were performed using peptides labelled with stable isotopes (13C and 15N) as internal standards. Fifteen food samples containing rabbit meat and/or liver were analysed to verify compliance of the rabbit meat and liver composition with product labelling. One sample was adulterated with undeclared rabbit liver. The limit of detection and limit of quantification for the selected peptides of interest were in the range of 0.17 to 0.35 ng/mg and 0.57 to 1.17 ng/mg, respectively. The method may be useful for the determination of rabbit meat and liver tissue in highly processed food samples.

LC-MS/MS-based quantification of tryptophan, kynurenine, and kynurenic acid in human placental, fetal membranes, and umbilical cord samples.

Tryptophan breakdown metabolites formed along the kynurenine pathway play a significant role in pregnancy and fetal development. To understand their involvement, it is crucial to quantify the levels of tryptophan (TRP), kynurenine (KYN), and kynurenic acid (KYNA) in relevant biological samples such as the placenta, fetal membranes, and umbilical cord. This study used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine TRP, KYN, and KYNA levels. The LC-MS/MS method was optimized for high sensitivity and specificity, demonstrating good reproducibility with a precision of < 10% CV and an accuracy of 85-115%. The lower limit of quantification for both TRP and KYN was 0.5 µg/ml, while for KYNA, it was 0.5 ng/mL. The method exhibited linearity within the examined range of concentrations in the homogenate, ranging from 0.5 to 30 µg/ml for TRP and KYN and from 0.5 to 25 ng/ml for KYNA. Using this method, we found significant differences in the concentrations of these substances in investigated maternal-fetal compartments. Placenta samples exhibited higher KYN and lower KYNA concentrations than the umbilical cord and fetal membrane, indicating a potentially important role for kynurenines in late pregnancy. Collectively, this finding may facilitate further research and provide inside into the involvement of the kynurenine pathway of TRP metabolism in fetal development.

LC-MS Metabolomic Profiling of Five Types of Unrefined, Cold-Pressed Seed Oils to Identify Markers to Determine Oil Authenticity and to Test for Oil Adulteration.

The authenticity of food products marketed as health-promoting foods-especially unrefined, cold-pressed seed oils-should be controlled to ensure their quality and safeguard consumers and patients. Metabolomic profiling using liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QTOF) was employed to identify authenticity markers for five types of unrefined, cold-pressed seed oils: black seed oil (Nigella sativa L.), pumpkin seed oil (Cucurbita pepo L.), evening primrose oil (Oenothera biennis L.), hemp oil (Cannabis sativa L.) and milk thistle oil (Silybum marianum). Of the 36 oil-specific markers detected, 10 were established for black seed oil, 8 for evening primrose seed oil, 7 for hemp seed oil, 4 for milk thistle seed oil and 7 for pumpkin seed oil. In addition, the influence of matrix variability on the oil-specific metabolic markers was examined by studying binary oil mixtures containing varying volume percentages of each tested oil and each of three potential adulterants: sunflower, rapeseed and sesame oil. The presence of oil-specific markers was confirmed in 7 commercial oil mix products. The identified 36 oil-specific metabolic markers proved useful for confirming the authenticity of the five target seed oils. The ability to detect adulterations of these oils with sunflower, rapeseed and sesame oil was demonstrated.

Heat-stable peptide markers specific to rabbit and chicken liver tissue for meat product authentication testing.

A three-step analysis was used to detect and identify heat-stable peptide markers specific to liver tissue from rabbit and chicken. It involved peptide discovery by liquid chromatography coupled to high resolution mass spectrometer (LC-HRMS), followed by protein identification using Spectrum Mill software and multiple reaction monitoring (MRM) based confirmation of the discovered peptides using a liquid chromatography coupled to triple quadrupole mass spectrometer (LC-TQ). We identified 50 and 91 heat-stable peptide markers unique to chicken and rabbit liver, respectively. The markers were validated in commercial food samples with declared liver tissue contents ranging from 5% to 30%. The best candidate peptides for distinguishing liver tissue from skeletal muscle were selected and then confirmed using MRM-based approach. Limit of detection of liver was found to be in the range of 0.13 to 2.13% (w/w) for chicken liver-specific peptide markers, and from 0.04 to 0.6% (w/w) for rabbit liver-specific peptide markers.

Identification of sunflower, rapeseed, flaxseed and sesame seed oil metabolomic markers as a potential tool for oil authentication and detecting adulterations.

Testing the composition, quality and authenticity of edible oils is crucial to safeguard the consumers' rights and health. The aim of our study was to identify oil-specific markers to enable the differentiation and authentication of sunflower, sesame, flaxseed and rapeseed oils, and to evaluate their antioxidant activity, total phenolic and carotenoid content. A metabolomic approach based on liquid chromatography coupled to quadrupole-time-of-flight mass spectrometry was employed for marker discovery. Spectrophotometric method was used for determination of antioxidant activity, total phenolic and carotenoid content. 76 oil samples from the four different manufacturers were examined. We identified 13 oil-specific markers for sunflower seed oil, 8 for rapeseed oil, 5 for sesame seed oil and 3 for flaxseed oil, their retention times, accurate masses, and characteristic fragment ions are reported. The abundances of the markers for each plant species were found to vary depending on the oil producer and the product batch. Significant differences in antioxidant activity, total phenolic and carotenoid content were also observed both between oils and within oil type. The highest total phenolic content (84.03 ± 4.19 to 103.79 ± 3.67 mg of gallic acid/kg) and antioxidant activity (245.67 ± 7.59 to 297.22 ± 2.32 mg Trolox/kg) were found in sesame seed and flaxseed oils, respectively. Identified metabolic markers can be used as qualitative markers to confirm the authenticity or to detect adulterations of oils. Composition, properties and authenticity testing should be more rigorous for food products marketed as health-promoting.

Comparison of the Determination of Some Antihypertensive Drugs in Clinical Human Plasma Samples by Solvent Front Position Extraction and Precipitation Modes.

The determination of the selected antihypertensive drugs in human plasma samples with the novel solvent front position extraction (SFPE) technique is presented. The SFPE procedure combined with LC-MS/MS analysis was used for the first time to prepare a clinical sample containing the drugs mentioned above from different therapeutic groups. The effectiveness of our approach was compared with the precipitation method. The latter technique is usually used to prepare biological samples in routine laboratories. During the experiments, the substances of interest and the internal standard were separated from other matrix components using a prototype horizontal chamber for thin-layer chromatography/high-performance thin-layer chromatography (TLC/HPTLC) with a moving pipette powered by a 3D mechanism, which distributed the solvent on the adsorbent layer. Detection of the six antihypertensive drugs was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in multiple reaction monitoring (MRM) mode. Results obtained by SFPE were very satisfactory (linearity R2 ≥ 0.981; %RSD ≤ 6%; LOD and LOQ were in the range of 0.06-9.78 ng/mL and 0.17-29.64 ng/mL, respectively). The recovery was in the range of 79.88-120.36%. Intra-day and inter-day precision had a percentage coefficient of variation (CV) in the range of 1.10-9.74%. The procedure is simple and highly effective. It includes the automation of TLC chromatogram development, which significantly reduced the number of manual operations performed, the time of sample preparation and solvent consumption.

Pork liver tissue-specific peptide markers for food authenticity testing and adulteration detections.

A liquid chromatography-mass spectrometry bottom-up proteomic approach was applied for the detection and identification of proteins from liver tissues. We identified 74 unique pork liver peptide markers that are resistant to the thermal processing of food. These peptides are derived from 43 proteins, which perform various functions in the liver. Roasted and sterilised pâté-type products with a pork liver content ranging from 6% to 51% were examined to select the most reliable pork liver peptide markers that survive unmodified in complex processed food matrices. Of the 74 specific heat-stable peptides detected in pure liver tissue, five (GDAPEEEVSLSK, ALTAELEAVGK, TFYLNVLNEEER, AQFGQPEILLGTIPGTGGTQR and VIAPGFNALEQILQSTAGK) were the best candidates to confirm the presence of liver tissue in highly processed meat products. We have identified unique tissue-specific markers that enable rapid and specific identification of pork liver in processed food and may contribute to the development of new methods for testing food authenticity.

Reactions of 3-Hydroxy-2-phenyl-1H-benzo[e]isoindol-1-one: A Route to 3-Hydroxy-/3-anilinobenzo[e]indan-1-ones and Benzo[f]phthalazin-1(2H)-ones.

New hydroxy- and anilinoindanone derivatives 3 and 4 were synthesized starting from 3-hydroxybenzo[e]isoindolinone 1 via the addition of alkyllithium (s-BuLi, n-BuLi, MeLi or i-PrLi) to the carbonyl group, followed by lactam ring opening and, finally, an intramolecular cyclization leading to target compounds. The same starting material was used for the preparation of the new benzo[f]phthalazinone derivatives 12-16 through multi-step reactions. The target derivative 16 was obtained from the corresponding bromolactam 15 by the Buchwald-Hartwig amination. Structures of the obtained compounds were confirmed by the NMR spectra.

Polyphenols composition and the biological effects of six selected small dark fruits.

The health-promoting activities of fruits are in the limelight in view of the growing risks posed by civilisational diseases and are connected with polyphenols. The present study examined bilberry, blueberry, blackcurrant, redcurrant, cherry and plum for their polyphenolic content and biological activities. The contents of total polyphenolic compounds and their subclasses were determined. Liquid chromatography high-resolution mass spectrometry was used to characterise the polyphenolic profiles. Small dark fruits' antioxidant, anti-inflammatory, and anti-cholinesterase activities were also extensively determined. Significant qualitative and quantitative differences in the analysed fruits' polyphenols composition and biological activities were demonstrated. The highest polyphenolic contents and antioxidant activities were established in blackcurrant fruit, but bilberry also had our attention due to an additional mild influence on antioxidant enzymes. The condensed tannin content in small dark fruits is developed. All tested fruits exhibit anti-inflammatory and anti-cholinesterase activities.

Unexpected content of kynurenine in mother's milk and infant formulas.

Mother's milk is widely recommended as complete food for the offspring in earliest postnatal time. However, the knowledge about detailed composition and the physiological role of bioactive components of breast milk is incomplete. Therefore, the aim of our study was to determine the content of kynurenine (KYN) in human breast milk during lactation and to explore the effects exerted by intragastric KYN administration from birth to weaning on physical and psychomotor development of adult rats. We found that KYN is consistently present in human milk and its content gradually increased from day 4 to 28 after delivery and that it is present in commercial baby formulas in amounts noticeably exceeding its physiological range. Animal studies showed that KYN supplementation resulted in a marked elevation of absorptive surface of rat intestine and in enhanced expression of both, aryl hydrocarbon receptor and G protein-coupled receptor 35 in the intestinal tissue in rats. Moreover, we discovered that KYN administration from birth to weaning resulted in neurobehavioral changes in adult rats. Therefore, we postulate that further research is required to thoroughly understand the function of KYN in early developmental stages of mammals and to ensure the safety of its presence in baby food products.

Peptide markers for distinguishing guinea fowl meat from that of other species using liquid chromatography-mass spectrometry.

The inability to easily identify the animal species in highly processed meat products makes them highly susceptible to adulterations. Reliable methods for detecting the species origin of meat used in processed food are required to ensure adequate labelling and minimize food fraud and allergenic potential. Liquid chromatography high resolution mass spectrometry was employed to identify new heat-stable guinea-fowl-specific peptide markers that can differentiate guinea fowl meat from other commonly consumed animal species, including closely related poultry species, in highly processed food products. We identified 26 unique guinea-fowl-specific markers. The high stability of guinea-fowl-specific peptides was confirmed by analysing food products with guinea fowl meat content ranging from 4% to 100%. The findings indicate that sensitive and reliable LC-MS/MS methods can be developed for the targeted detection and quantification of guinea fowl meat in highly processed meat products.

Sotalol does not interfere with the antielectroshock action of selected second-generation antiepileptic drugs in mice.

BACKGROUND: Due to blocking ß-receptors, and potassium KCNH2 channels, sotalol may influence seizure phenomena. In the previous study, we have shown that sotalol potentiated the antielectroshock action of phenytoin and valproate in mice. MATERIALS AND METHODS: As a continuation of previous experiments, we examined the effect of sotalol on the action of four chosen second-generation antiepileptic drugs (oxcarbazepine, lamotrigine, pregabalin, and topiramate) against the maximal electroshock in mice. Undesired effects were evaluated in the chimney test (motor impairment) and step-through passive-avoidance task (long-term memory deficits). Finally, brain concentrations of antiepileptics were determined by fluorescence polarization immunoassay, while those of sotalol by liquid chromatography-mass spectrometry. RESULTS: Sotalol at doses of up to 100 mg/kg did not affect the electroconvulsive threshold. Applied at doses of 80-100 mg/kg, sotalol did not affect the antielectroshock action of oxcarbazepine, lamotrigine, pregabalin, or topiramate. Sotalol alone and in combinations with antiepileptics impaired neither motor performance nor long-term memory. Finally, sotalol significantly decreased the brain concentrations of lamotrigine and increased those of oxcarbazepine and topiramate. Pharmacokinetic interactions, however, did not influence the final antielectroshock effects of above-mentioned drug combinations. On the other hand, the brain concentrations of sotalol were not changed by second-generation antiepileptics used in this study. CONCLUSION: Sotalol did not reduce the antielectroshock action of four second-generation antiepileptic drugs examined in this study. Therefore, this antidepressant drug should not interfere with antiseizure effects of lamotrigine, oxcarbazepine, pregabalin, and topiramate in patients with epilepsy. To draw final conclusions, our preclinical data should still be confirmed in other experimental models and clinical conditions.

LIQUID CHROMATOGRAPHY-MASS SPECTROMETRY BOTTOM-UP PROTEOMIC METHODS IN ANIMAL SPECIES ANALYSIS OF PROCESSED MEAT FOR FOOD AUTHENTICATION AND THE DETECTION OF ADULTERATIONS.

This review offers an overview of the current status and the most recent advances in liquid chromatography-mass spectrometry (LC-MS) techniques with both high-resolution and low-resolution tandem mass analyzers applied to the identification and detection of heat-stable species-specific peptide markers of meat in highly processed food products. We present sets of myofibrillar and sarcoplasmic proteins, which turned out to be the source of 105 heat-stable peptides, detectable in processed meat using LC-MS/MS. A list of heat-stable species-specific peptides was compiled for eleven types of white and red meat including chicken, duck, goose, turkey, pork, beef, lamb, rabbit, buffalo, deer, and horse meat, which can be used as markers for meat authentication. Among the 105 peptides, 57 were verified by multiple reaction monitoring, enabling identification of each species with high specificity and selectivity. The most described and monitored species by LC-MS/MS so far are chicken and pork with 26 confirmed heat-stable peptide markers for each meat. In thermally processed samples, myosin, myoglobin, hemoglobin, l-lactase dehydrogenase A and ß-enolase are the main protein sources of heat-stable markers. © 2019 John Wiley & Sons Ltd. Mass Spec Rev.

LC-QTOF/MS determination of tryptophan and kynurenine in infant formulas.

A rapid, reliable and sensitive liquid chromatography quadrupole time-of-flight mass spectrometry method for the determination of tryptophan and its metabolite kynurenine in milk formulas for neonates and infants was developed and validated. Two extraction techniques based on EMR Lipid QuEChERS and liquid-liquid extraction with diethyl ether to extract lipids and methanol to precipitate the protein were tested and compared. Four different infant formula products were randomly selected and evaluated for the effect of co-extracted matrix components on the quantitative analysis results. The influence of matrix components on analytical signals was normalized by the use of stable isotope-labeled standards and matrix-matched calibration. The developed method was found to be sensitive and effective for both analytes in all the examined infant formulas with satisfactory linearity (R2 ≥ 0.9995), recovery in the range of 75.7% ± 4.5 - 99.0% ± 1.1, and intra- and inter-day precision with the coefficient of variation below 6.3% and 17.9%, respectively. The limits of detection (LOD) and quantification (LOQ) for both compounds differed significantly between the examined formulas. The LOD and LOQ values were found to be in the range of 2.18-9.85 µg/g and 6.61-29.84 µg/g for the determination of tryptophan and in the range of 0.21-2.71 µg/g and 0.63-8.23 µg/g for the determination of kynurenine, respectively. The method was proved to be suitable for the determination of tryptophan and kynurenine in infant formulas, and it can be used to study the link between tryptophan metabolism via kynurenine pathway and metabolic disorders in infants.

LC-QTOF-MS identification of rabbit-specific peptides for authenticating the species composition of meat products.

Rabbit is a healthy meat, with low allergenicity and excellent nutritional properties. The global popularity of rabbit meat makes it a target for food fraud. We present a LC-QTOF-MS/MS approach for detecting and identifying rabbit-specific peptide-markers from thermally processed meat products to differentiate rabbit from other commonly-consumed animal species. We identified 49 heat-stable specific peptides. We selected the most stable markers for testing complex meat matrices by analysing pâtés-type products with a rabbit meat content ranging from 5% to 85%. Of the 49 heat-stable peptides detected in pure cooked rabbit meat, three were consistently detected in all investigated pâté samples i.e., SSVFVADPK, AFFGHYLYEVAR and PHSHPALTPEQK. Monitoring meat species other than rabbit in the examined pâtés using pork-, lamb- and chicken-specific peptides identified the presence of undeclared chicken in two samples. The results confirm that LC-QTOF-MS/MS is a suitable tool for multi-species detection in processed meat products, particularly for authentication purposes.

Pesticides and conservation of large ungulates: Health risk to European bison from plant protection products as a result of crop depredation.

The coexistence of large mammals and humans in the contemporary landscape is a big challenge for conservationists. Wild ungulates that forage on arable fields are exposed to the negative effects of pesticides, and this problem also applies to protected species for which intoxication by pesticides may pose a health risk and directly affect the effectiveness of conservation efforts. In this paper we assessed the threat posed by pesticides to the European bison Bison bonasus, a species successfully restituted after being extinct in the wild. We studied samples of B. bonasus liver from three free-living populations in Poland (Bialowieska, Knyszynska, and Borecka forests) and captive individuals from breeding centres. LC-QTOF-MS/MS two-step analysis for the detection, identification and confirmation of pesticide residues in liver samples, which included MS and targeted MS/MS scans, was conducted. It was found that European bison are exposed to pesticides as a result of crop depredation: the presence of tetraconazole, fluopyram and diazinon residues in 12 liver samples was confirmed. The concentration levels of the detected substances were quite low, but in the liver samples more than one substance was usually found, and the potential health risk to European bison may result from the synergistic interaction of these substances. The place of occurrence of the population, abundance, and the management regime affect the exposure of European bison to pesticides. Due to the high conservation status of the European bison, the monitoring of intoxication by pesticides should be included in the conservation plans of this species. This issue should also be more widely included in the study of other wild ungulates because knowledge about the impact of pesticides on wildlife is still insufficient.

LC-MS/MS determination of pesticide residues in fruits and vegetables.

The aim of the research is to evaluate pesticide residue contamination of fresh and frozen fruits and vegetables, agricultural raw material, purchased from Polish farmers for production of frozen fruits and vegetables, and the estimation of the multiresidue method effectiveness expressed as the proportion of pesticides detected in food samples to the total number of pesticides analyzed by multiresidue methods. A total of 144 samples (of black currants, red currants, raspberries, cherries, strawberries, blackberries, cauliflowers and broccoli) were analyzed using LC-MS/MS method for the determination of 60 pesticides. QuEChERS extraction, matrix-matched calibration and dynamic multiple reaction monitoring method were used. Residues of 15 compounds, mainly fungicides and insecticides, were detected in 46 samples. The percentage of samples with residues above the maximum residue levels (MRL) was 15%, whereas samples with residues below MRL were 17%. A total of 13 samples contained more than one pesticide residue. Pesticide residues were detected most often in samples of black currants (50%), broccoli (36.4%), raspberries (29%) and red currants (21.8%). The most frequently detected pesticides were carbendazim and acetamiprid. The proportion of pesticides detected during our study to the total number of analyzed pesticides amounted to 25%. It was compared to literature findings. For three fourth of multiresidue methods, the proportion was below 50% for methods developed for the analysis of less than 100 pesticides, and below 30% for methods developed for the analysis of more than 100 pesticides. It appears that a lot of efforts and means is lost on pesticides never or rarely detected in examined samples. The workload and cost effectiveness of the development and application of multiresidue methods along with the range of pesticides covered by the method should be carefully and thoroughly considered anytime when a new method or workflow is developed. Including non-targeted screenings in pesticide residue control seems to be an alternative worth considering.

Study on vitamin D2 stability in dried mushrooms during drying and storage.

The main objective of this work was to determine the stability of vitamin D2 in dried mushrooms Agaricus bisporus, Pleurotus ostreatus and Lentinula edodes during storage, as well as to examine the possibility of inducing vitamin D2 production in dried mushrooms by UVB irradiation. After 1.5 year storage of dried mushrooms, the level of vitamin D2 in button mushrooms was found to be 6.90 µg/g dw, which is a 48.32% of initial level of vitamin D2. In the case of dried oyster and shiitake mushrooms there was a decrease to the level of 66.90% and 68.40%, respectively. It was determined that dried mushrooms can produce ergocalciferol under UVB irradiation. The highest content of vitamin D2 was observed in A. bisporus. Freeze-dried A. bisporus contained from 42.08 to 119.21 µg/g dw and hot-air dried mushrooms contained from 21.51 to 81.17 µg/g dw vitamin D2.