Tandem Mass Spectrometric Detection Research Articles

Stability and degradation mechanism of (−)-epicatechin in thermal processing

(−)-Epicatechin (EC) is a commonly dietary phytochemical that presents multi-physiological activities on human health. Thermal processing is a common method to extract EC, albeit likely to degrade EC considering its thermal instability. In this study, an 85-min non-durable bathing incubation assay was designed to simulate the state of EC in boiling water while cooking. Monitoring of degradation products was performed using ultra-performance liquid chromatography combined with electrospray ionization quadrupole tandem mass spectrometric detection (UPLC-ESI-TSQ-MS/MS). The results revealed that ca. 65.2 % loss of EC was detected in the first 10 min, and over 99.5 % of EC was degraded within 30 min. A total of 22 degradation products were identified based on retention time, full and tandem MS data were the first to be comprehensively reported. Isomerization, oxidation, hydroxylation, dimerization, and ring cleavage were the main chemical reactions that occurred for EC in boiling aqueous solution.

Bioequivalence study between two formulations of betamethasone dipropionate and betamethasone disodium phosphate injectable suspension in healthy adults

Objective: To evaluate bioequivalence between two formulations of 5,0 mg/mL betamethasone dipropionate + 2,0 mg/mL betamethasone disodium phosphate injectable suspension in healthy adults under fasting condition. Methods: The study was an open label, randomized, single dose, 2x2 crossover study in 36 healthy adult subjects under fasting conditions. Betamethasone concentrations in human plasma were determined using a validated liquid chromatography coupled to a tandem mass spectrometer detector method. Results: Statistical analysis has determined confidence intervals, power of the test and p-value for sequence effect to the pharmacokinetic parameters Cmax, AUC0-t and AUC0-∞. The geometric mean ratio (90%CI) of the test drug/reference drug for betamethasone were 87.74% to 92.23% for Cmax, 93.95% to 98.91% for AUC0-t and 94.36% to 99.95% for AUC0-∞. Power of the test was 100% for all parameters and p-value for sequence effect were 33.39% for Cmax, 19.98% AUC0-t, and 24.32% for AUC0-∞. Conclusion: Reference and test formulations are statistically bioequivalent and, therefore, interchangeable, according to the local and international criteria, since confidence intervals for Cmax and AUC0-t ratios were within 80% and 125%, according to Anvisa resolution RE nº 1170/2006.

Study of chili peppers and paprika contamination with alternaria toxins as health risk factors

At present, researchers show considerable interest in unregulated toxins of widespread fungi in nature, in particular, metabolites of microfungi of Alternaria genus. These toxins are potentially hazardous for human health and, under unfavorable conditions, capable of producing toxic metabolites, Alternaria toxins (AT), which have shown genotoxicity, mutagenicity and acute toxic effects. They are frequently detected not only in cereals and oilseeds, fruits, nuts, vegetables, but also in spices, especially dried red peppers. The aim of the study was to investigate the occurrence of Alternaria toxins in paprika and chili peppers sold on the domestic market as well as to assess their intakes by humans through their consumption. Concentration of 5 Alternaria toxins (alternatiol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), tenuazonic acid (TeA)) was detected in 37 samples of dried red peppers marketed in Moscow and Moscow region in 2024 year including paprika (20 samples) and chili pepper (17 samples). The analysis of Alternaria toxins contamination was carried out by high-performance liquid chromatography coupled to tandem mass-spectrometric detection (HPLC-MS/MS). TeA was detected in 84 % of red pepper samples in concentration from 43 to 3295 µg/kg and TEN – in 40 % cases, in low levels ranging from 1.0 to 11 µg/kg. The occurrence of AT in paprika was higher than in chili pepper. Combined (2 or more toxins) contamination with Alternaria toxins was found only in paprika samples (65 %; the predominant combination is TeA and TEN). Intakes of Alternaria toxins associated with consumption of chili and paprika did not exceed the reference values and ranged from 0.0003 % (for TEN) to 0.24 % (for TeA) of the threshold of toxicological concern (TTC).

Результаты оценки контаминации перца чили и паприки альтернариатоксинами как факторы риска здоровью

At present, researchers show considerable interest in unregulated toxins of widespread fungi in nature, in particular, metabolites of microfungi of Alternaria genus. These toxins are potentially hazardous for human health and, under unfavorable conditions, capable of producing toxic metabolites, Alternaria toxins (AT), which have shown genotoxicity, mutagenicity and acute toxic effects. They are frequently detected not only in cereals and oilseeds, fruits, nuts, vegetables, but also in spices, especially dried red peppers. The aim of the study was to investigate the occurrence of Alternaria toxins in paprika and chili peppers sold on the domestic market as well as to assess their intakes by humans through their consumption. Concentration of 5 Alternaria toxins (alternatiol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), tenuazonic acid (TeA)) was detected in 37 samples of dried red peppers marketed in Moscow and Moscow region in 2024 year including paprika (20 samples) and chili pepper (17 samples). The analysis of Alternaria toxins contamination was carried out by high-performance liquid chromatography coupled to tandem mass-spectrometric detection (HPLC-MS/MS). TeA was detected in 84 % of red pepper samples in concentration from 43 to 3295 µg/kg and TEN – in 40 % cases, in low levels ranging from 1.0 to 11 µg/kg. The occurrence of AT in paprika was higher than in chili pepper. Combined (2 or more toxins) contamination with Alternaria toxins was found only in paprika samples (65 %; the predominant combination is TeA and TEN). Intakes of Alternaria toxins associated with consumption of chili and paprika did not exceed the reference values and ranged from 0.0003 % (for TEN) to 0.24 % (for TeA) of the threshold of toxicological concern (TTC).

Identification and formation of key aroma-active compounds in red jujube as affected by different processing methods

Processing methods significantly affect the aroma characteristics of red jujube; however, few studies have explored how. Gas chromatography–tandem mass spectrometric detection, gas chromatography–olfactometry, relative odor activity value and quantitative descriptive analysis were used to investigate the aroma changes of red jujube during typical processing (freeze drying, baking, frying and steaming); furthermore, the formation of aroma-active compounds in processed red jujube was predicted based on untargeted metabolomics. After freeze drying, the total aroma content increased by 0.90 %, while it decreased by 51.59 %, 74.11 % and 78.74 % after baking, frying and steaming, respectively. In addition, ethyl esters dominated the sweet and fruity notes in freeze-dried red jujube, which were formed via combination of fatty acids metabolism, pyruvate metabolism and amino acid catabolism. (E,E)-2,4-Decadienal derived from fatty acid oxidation dominated the fatty note in fried red jujube. 2-Ethyl-3,5-dimethylpyrazine formed via the Maillard reaction dominated the roasty notes of baked red jujube.

Ecofriendly UPLC-MS/MS method for simultaneous assay of veterinary drugs residues; erythromycin, sulfadiazine and trimethoprim in edible chicken tissues, evaluation of method greenness and whiteness

Antibiotics are the key veterinary drugs used in animal food production. The uncontrolled widespread use of antibiotics may lead to the accumulation of their residues in foodstuff such as milk, eggs, meat and muscles. An Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS/MS) method was developed for simultaneous determination of triple combination of erythromycin thiocyanate (ERY), sulfadiazine sodium (SFD) and trimethoprim (TMP) in their dosage form and in different chicken tissues including muscles and liver samples. A Simple extraction and cleaning up procedure was applied using 0.1 N EDTA and methanol to remove the interfering substances and hence increase the efficiency of chromatographic separation. Moreover, separation was performed within 1.2 min on Agilent Poroshell 123EC-C18 column using a mobile phase mixture of methanol: water (90:10, v/v), at a flow rate of 0.5 mL/min using quadrupole tandem mass spectrometer detector. The method was validated according to Food and Drug Administration (FDA) guidelines and linearity was achieved in the range of 2.5–1000 ng/mL for ERY and TMP and in the range of for 5–2000 ng/mL for SFD using tamsulosin (TAM) as an internal standard. All the validation results were within the acceptable ranges. Moreover, Three green metric systems including Green Analytical Procedure Index (GAPI), Analytical Eco-Scale and Analytical GREEnness (AGREE) were used to assess the greenness of the developed method. As well as, the novel blue applicability grade index (BAGI) tool was applied to evaluate the practicality of the method. Results of these tools proved the low environmental impact of the method regarding solvent consumption, analysis time, amount of sample needed, sample treatment procedure, and waste product. The developed method has the privilege of high sensitivity and selectivity as well as short analysis time; hence it can be used to test the presence of the residues of studied drugs in various marketed meat samples to assure human safety and maintain public health. Moreover, it is the first developed method for their analysis in liver samples.

Bioequivalence of zolpidem hemitartrate: new formulations of 5 mg SL, 10 mg and 12.5 mg XR and comparison of their bioavailabilities

Objective: To evaluate pharmaceutical bioequivalence between two formulations of 5 mg zolpidem hemitartrate sublingual and, 10 and 12.5 mg extended-release formulations tablets in healthy subjects under fasting and fed conditions. Methods: An open label, monocentric, randomized, 2 x 2 crossover study in 40 healthy adults under fasting conditions comparing two formulations of zolpidem 5mg sublingual tablets. Analyte concentrations in human plasma were determined using a validated liquid chromatography with a tandem mass spectrometer detector method (LC-MS/MS). The same design was utilized to evaluate the other formulations.
 Results: Statistical analysis has determined geometric mean of test / reference ratio, confidence intervals, and power of the test to the pharmacokinetic parameters Cmax, AUC0-t, and AUC0-∞. The geometric mean ratio (90%CI) of the test drug/reference drug for zolpidem 5mg were 99.89 to 113.57% for Cmax, 97.15% to 108.40% for AUC0-t, and 97.22% to 108.13% for AUC0-∞. Power of the test was 99.35% for Cmax and 100% AUC0-t, and AUC0-∞. Conclusion: Both test and reference are bioequivalent for all formulations and, therefore, they are interchangeable, according to the Brazilian criteria (Anvisa resolution RE nº 1170/2006), since confidence intervals for Cmax and AUC0-t ratios were within 80% and 125%.

Diagnosis of artificial hypoglycemia due to oral hypoglycemic drugs by high-performance liquid chromatography with tandem mass spectrometric detection

BACKGROUND: Artificial hypoglycemia (ArH) is a decrease of blood glucose levels less than 3 mmol/l due to the deliberate use of hypoglycemic drugs by a patient outside of medical appointments. Timely diagnosis of this kind of hypoglycemia avoids unnecessary numerous examinations and hospitalizations. However, the detection of ArH still remains an extremely difficult task for both the healthcare facility and the attending physician. The foreign literature describes cases of successful detection of deliberate intake of oral hypoglycemic drugs (OHD) using high-performance liquid chromatography with tandem mass spectrometric detection (HPLC-MS/MS). Thus, it is relevant to develop and validate a method for determining OHD using HPLC-MS/MS.AIM: To optimize the diagnosis of ArH due to the use of OHD.MATERIALS AND METHODS: A total of 92 patients were examined. The development of the HPLC-MS/MS method for the detection of the studied OHD (sOHD; n=1-glibenclamide, n=1-gliquidone, n=1-gliclazide, n=1-glimepiride, n=1-glipizide, n=1-nateglinide and n=1-repaglinide) in the blood was carried out in a group of patients with diabetes mellitus type 2 (n=7) who received sOHD, and a group of conditionally healthy people who did not receive any medications (n= 7). To validate the method, the determination of sOHD substances was carried out on groups of patients with hyperinsulinemic nondiabetic hypoglycemia (NDH) of unknown origin (n=11) and with insulinoma (n=67).RESULTS: In the study of blood samples by HPLC-MS/MS in the group of patients with diabetes mellitus type 2, was confirmed in 100% of the cases the use of the drug that the patient received, in the group of conditionally healthy — sOHD were not detected. A false positive result was not obtained in any conditionally healthy and in any patient with insulinoma. ArH was diagnosed in 5 out of 11 patients in the group with hyperinsulinemic NDH of unknown origin, the method identified sOHD glibenclamide and gliclazide in the patients’ blood samples. In the remaining 6 patients of this group, examinations were continued and other causes of NDH were diagnosed. The sensitivity of the method was 100% [74%; 100%], specificity — 100% [95%; 100%].CONCLUSION: The HPLC-MS/MS method has high diagnostic accuracy in the detection and identification of sOHD (glibenclamide, gliquidone, gliclazide, glimepiride, glipizide, nateglinide and repaglinide) in blood samples of patients receiving these drugs. Currently, due to the low availability of the method, this study is advisable to use in patients with hyperinsulinemic hypoglycemia with negative results of first-line insulinoma imaging methods (computed tomography with contrast enhancement, ultrasound and magnetic resonance imaging of the abdominal cavity).

Simple and Practical Method for the Quantitative High-Sensitivity Analysis of N-Nitroso Duloxetine in Duloxetine Drug Products Utilizing LC-MS/MS.

The high-sensitivity analytical method for the determination of N-nitroso duloxetine (NDXT), which can be carcinogenic and harmful in duloxetine drug products, was successfully developed utilizing liquid chromatography-tandem mass spectrometry (LC-MS/MS). Tandem mass spectrometric detection at positive electrospray ionization in multiple reaction monitoring (MRM) mode was then employed for the determination of NDXT. The quantitative range for NDXT was found in 0.075-3.75 ng/mL in terms of concentration in the dilution solvent for duloxetine active pharmaceutical ingredient (API) and capsules and 0.075-1.875 ng/mL for duloxetine tablets, and the recovery rates were in the range of 82.5-91.6% for the API, 91.0-113.4% for capsules, and 70.6-109.1% for tablets, respectively. The repeatability was 6.9% with a %RSD of n = 9 for the API, 10.9% with a %RSD of n = 9 for capsules, and 21.6% with a %RSD of n = 9 for tablets, respectively. For reproducibility, the %RSD of the n = 6 measurements between the two sites was 3.5%. The calibration curve of NDXT in the concentration range of 0.075-3.75 ng/mL was carried out, and the correlation coefficient (R) was found to be 1.000. The sample solution was stable for 7 days. The applicability of the determination of the content of NDXT in a variety of duloxetine drug products was demonstrated. This manuscript seeks to aid the risk assessment process of NDXT in duloxetine drug products through providing a fast and reliable quantitative LC-MS/MS analytical method.

High-resolution mass spectrometric elucidation of electron ionization induced fragmentation pathways of methylated warfarin and selected hydroxylated species

The plant secondary metabolite families of coumarin and 4-hydroxy coumarin have a broad pharmacological spectrum ranging from antibacterial to anticancer properties. One prominent member of this substance class is the synthetic but naturally inspired anticoagulant drug and rodenticide warfarin (coumadin). A vast number of publications focus on the identification of warfarin and its major cytochrome P450-mediated phase I metabolites by liquid chromatography (LC) with mass spectrometry (MS) and tandem mass spectrometric (MS/MS) detection techniques. For the first time, electron ionization (EI) induced high-resolution quadrupole time-of-flight mass spectrometric (HR-qToF-MS) data of in-liner derivatized warfarin and selected hydroxylated species is provided in this study as an alternative to LC-MS/MS approaches. Furthermore, the characteristic fragments and fragmentation pathways of the analyzed methyl ethers are concluded. The obtained data of analytical standards, specific deuterated and 13C-labeled compounds prove inductive cleavage of the acyl or acetonyl side chain, methyl migration, and H-migration, along with consequential inductive cleavage as predominant fragmentation routes. Based on the HR-spectral data, commonalities and differences between the analyzed compounds and fragment groups were evaluated with future applicability in structure elucidation and spectra prediction of related compounds.

Implementation of the biomonitoring system in large scientific expeditions on the example of the scientific and educational project "Arctic Floating University"

Currently, biomonitoring research is actively developing in the Arctic region, despite this, groups of people who temporarily come to the Arctic remain outside the national monitoring system, unlike the local population. Participation in expeditions on ships implies special living conditions, such as limited space, sensory deprivation. The study aims to analyse the biochemical parameters of blood, the content of essential, toxic elements, persistent organic pollutants (POPs) and vitamin D in samples of the expedition group temporarily coming to work in extreme Arctic conditions. Scientists carried out a study on board the Mikhail Somov scientific expedition vessel as part of the Arctic Floating University project. The participants were 50 people: 26 men and 24 women aged 20 to 72 years. As a result of the analysis, the authors have determined: 8 biochemical blood parameters, vitamin D levels by high-performance liquid chromatography with tandem mass spectrometric detection, the content of essential (Co, Mn, Se, Cu, Zn), toxic elements (As, Cd, Hg, Pb) using an inductively coupled plasma mass spectrometer and POPs by gas chromatography-mass spectrometry. Almost 90% of the expedition participants had vitamin D concentrations below 20 ng/ml, which indicates a deficiency of this vitamin in the body. After conducting a comparative analysis between the expedition group and the permanent population of the Nenets Autonomous Okrug (NAO), it was found that the expedition group significantly reduced the content of not only toxic compounds (As, Cd, Hg, Pb, POPs), but also essential elements (Co, Mn, Se, Cu, Zn), according to compared to the local population. It is worth noting that the concentration of arsenic in the expedition group is 65 times lower (0.12 µg/L) than in permanent residents of the Arctic zone of the Russian Federation (7.29 µg/L), and the concentration of lead in the group of expedition workers was 42 times lower (0.30 µg/L) than in the local population (15.21 µg/L). The data obtained provide information about the elementary and toxic status of the urban population of the Russian Federation. One of the reasons for such differences may be the predominantly traditional lifestyle of the local NAO population and the biomagnification of toxic substances in food chains. If biomonitoring studies are carried out by specialists on an ongoing basis and the data obtained is accumulated, then a basis may appear for predicting risks of both a short-term and long-term nature. The conducted research contributes to the expansion of the biomonitoring system at the national level.

Alternaria toxins in tomato products marketed in the Russian Federation

Tomatoes and tomato products are widely produced and consumed throughout the world. Alternaria spp. are the main cause of alternariosis (black mold disease) on fresh tomatoes, both in the field and after harvesting. Alternaria toxins are widespread contaminants of tomato products. The aim of the present study was to evaluate the contamination of tomato processing products from the domestic market with Alternaria toxins, as well as to assess their intake by humans through the consumption of tomato juices. Material and methods. The content of Alternaria toxins (alternatiol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid) was determined in 64 samples of tomato products (paste, ketchup, juice) by high-performance liquid chromatography coupled to tandem mass-spectrometric detection (HPLC-MS/MS). Results. The priority Alternaria toxins for tomato paste, ketchup and juice were tenuazonic acid (61% of 64 samples, in amounts from 20.0 to 1065.5 μg/kg), altenuene (52%, 8.9-200.1 μg/kg) and alternariol (27%, 12.2-561.6 μg/kg). Samples of tomato paste turned out to be the most contaminated with Alternaria toxins while tomato juice samples were the least contaminated. At the same time, several toxins were found in 91% of tomato paste samples, 35% of ketchups, and 23% of tomato juices. Conclusion. To the best of our knowledge, the present study is the first survey devoted to Alternaria toxins contamination of tomato paste, ketchup and tomato juice sold on the Russian market. The high frequency of their contamination with tenuazonic acid, altenuene and, to a lesser extent, alternariol has been established, which indicates a potential risk to human health when tomato processing products are consumed. This indicates the need for a hygienic assessment of contamination the above products with tenuazonic acid, altenuene and alternariol. When calculating the potential intake of Alternaria toxins for different age population groups, it was shown that high levels of alternariol (up to 56.77 ng/kg body weight per day) could be obtained under daily consumption of tomato juice by adults and children under three years of age, as well as tenuazonic acid when consuming tomato juice contaminated at the 95th percentile level as part of the diet in organized groups for orphans and children without parental care.

Analysis of pesticide residues in potato varieties using modified quechers multi‐residue method combined with gas chromatography tandem mass spectrometric detection

AbstractRegular monitoring of pesticide residues in potatoes (Solanum tuberosum L.) is important to ensure a low risk of pesticide toxicity to consumers. An efficient, modified quick easy cheap effective rugged safe‐dispersive‐solid‐phase extraction (QuEChERS‐ d‐SPE) was optimized and validated for quantification of pesticide residues (metalaxyl, chlorpyrifos, λ‐cyhalothrin, and cypermethrin) in popular Kenyan potato varieties “Shangi,” “Tigoni,” and “Dutch Robyjn,” grown under controlled field conditions using gas chromatography with tandem mass spectrometry (GC‐MS/MS). An evaluation of the effect of five spraying frequencies (unsprayed, 5, 7, 10, and 14‐day intervals) of metalaxyl‐based fungicides on residue levels in harvested potatoes was simultaneously done. Metalaxyl residue levels in the varieties were determined for each spraying regime. Method validation showed good linearity (r2) of 0.988–0.999, detection limit (0.37–0.61 µg/L), quantification limit (1.20–1.84 µg/L) while recovery (71%–94%), and precision (relative standard deviation of 2.3%–24.1%) were used to determine the accuracy of the method. Pyrethroids (cypermethrin and λ‐cyhalothrin) and organophosphates (metalaxyl and chlorpyrifos) were detected in the potato samples. Residue levels of the compounds were not significantly different (p = 0.05) between varieties except for chlorpyrifos. Cypermethrin was the residue compound detected at the highest mean level with 17.4 µg/L, followed by metalaxyl, chlorpyrifos, and λ‐cyhalothrin, at 2.88, 1.05, and 0.15 µg/L, respectively. Residue levels at different spraying frequencies varied statistically between varieties (p = 0.05). Dutch Robyjn sprayed at seven days’ intervals had the highest residue level of 9.49 ± 0.26 µg/L. QuEChERS‐d‐SPE with GC‐MS/MS has shown suitability for routine pesticide residue monitoring in potatoes. The potatoes grown under the controlled experimental conditions did not present any concern for human health, as the pesticide residues detected were below Codex maximum residue levels of 500 µg/L.

Plasma oxidative lipidomics reveals signatures for sepsis-associated acute kidney injury

BackgroundOxidized lipids are essential bioactive lipid mediators generated during infection that regulate oxidative stress and the inflammatory response, but their signatures in patients with sepsis-associated acute kidney injury (SA-AKI) are poorly understood. This study analyzed the oxidative lipidomics of plasma from patients with SA-AKI to reveal the underlying biomarkers and pathophysiological mechanisms involved in sepsis. MaterialsA total of 67 patients with SA-AKI and 20 age- and sex-matched healthy controls (HCs) participated in this prospective cohort study. Among the patients with SA-AKI, 14 cases had stage I-II AKI and 53 cases had stage III AKI. Oxidative lipidomic analysis of plasma samples was conducted using ultra performance liquid chromatography coupled with tandem mass spectrometric (UPLC-MS /MS) detection. ResultsAmong 21 kinds of differentially oxidized lipids, 5(S),12(S)-DiHETE, 5-isoPGF2VI, 5,6-DiHETrE, 11,12-EET and 9,10-DiHOME showed the best performance. The prediction model incorporating them has shown highly sensitive and specific in distinguishing different stages of SA-AKI from HCs. The annotation of Kyoto Encyclopedia of Genes and Genomes illustrated that the overall downregulation of vascular smooth muscle contraction was closely related to the pathophysiological mechanism of SA-AKI. ConclusionThis study revealed alterations in the characteristic oxidized lipids in the plasma of SA-AKI patients, and these lipids had high diagnostic efficiency and potential targeted intervention value for SA-AKI.

Comparative metabolism of aflatoxin B1 in mouse, rat and human primary hepatocytes using HPLC–MS/MS

Aflatoxin B1 (AFB1) is a highly hepatotoxic and carcinogenic mycotoxin produced by Aspergillus species. The compound is mainly metabolized in the liver and its metabolism varies between species. The present study quantified relevant AFB1- metabolites formed by mouse, rat, and human primary hepatocytes after treatment with 1 µM and 10 µM AFB1. The use of liquid chromatographic separation coupled with tandem mass spectrometric detection enabled the selective and sensitive determination of phase I and phase II metabolites of AFB1 over incubation times of up to 24 h. The binding of AFB1 to macromolecules was also considered. The fastest metabolism of AFB1 was observed in mouse hepatocytes which formed aflatoxin P1 as a major metabolite and also its glucuronidated form, while AFP1 occurred only in traces in the other species. Aflatoxin M1 was formed in all species and was, together with aflatoxin Q1 and aflatoxicol, the main metabolite in human cells. Effective epoxidation led to high amounts of DNA adducts already 30 min post-treatment, especially in rat hepatocytes. Lower levels of DNA adducts and fast DNA repair were found in mouse hepatocytes. Also, protein adducts arising from reactive intermediates were formed rapidly in all three species. Detoxification via glutathione conjugation and subsequent formation of the N-acetylcysteine derivative appeared to be similar in mice and in rats and strongly differed from human hepatocytes which did not form these metabolites at all. The use of qualitative reference material of a multitude of metabolites and the comparison of hepatocyte metabolism in three species using advanced methods enabled considerations on toxification and detoxification mechanisms of AFB1. In addition to glutathione conjugation, phase I metabolism is strongly involved in the detoxification of AFB1.

Determination of 15 Functional State Biomarkers in Human Urine by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection

The capabilities and limitations of complex procedures for targeted metabolomic analysis using liquid chromatography in combination with tandem mass spectrometry (HPLC-MS/MS) are discussed. An HPLC-MS/MS procedure for the simultaneous determination of the concentrations of 15 biomarkers of the functional state of a human being in urine is presented. The target analytes are biogenic substances of various chemical natures, the basic concentrations of which in biomatrices can vary significantly, up to three orders of magnitude or more. Complex analysis is also difficult to perform due to significant differences in the hydrophilic and hydrophobic properties of the analytes. The testing of the procedure in a bioanalytical experiment made it possible to establish significant differences in the concentrations of a number of biomarkers in the urine of persons with different levels of physical fitness. With a high level of physical fitness, the concentrations of these compounds in urine have lower values in comparison with those in the control group.

Thermal degradation of (2R, 3R)-dihydromyricetin in neutral aqueous solution at 100 ℃

In the field of thermal degradation of flavonoids, current studies mainly focused on flavonols. However, the thermal degradation of dihydroflavonols in aqueous solution has received limited attention compared to flavonols. The single C2-C3 bonds of dihydroflavonols, which differs from the C2-C3 double bond in flavonols, may cause different degradation mechanisms. Dihydromyricetin (DMY) is a typical dihydroflavonol with six hydroxyl groups, and possesses various health effects. We explored the thermal degradation of DMY in neutral aqueous solution (pH 7) at 100 ℃. Ultra-performance liquid chromatography combined with photodiode array and electrospray ionization quadrupole-time-of-flight tandem mass spectrometric detection (UPLC-PDA-ESI-QTOF–MS/MS) provided suitable platform for exploring DMY degradation pathways, and negative ion mode was applied. Thermal treatment led to a decline in DMY level with time, accompanied by the appearance of various degradation products of DMY. Degradation mechanisms of DMY included isomerization, oxidation, hydroxylation, dimerization and ring cleavage. The pyrogallol-type ring B of DMY might be initially oxidized into ortho-quinone, which could further attack another DMY to form dimers. In addition, hydroxylation is likely to occur at C-2, C-3 of DMY or DMY dimers, which then further yields ring-cleavage products via breakage of the O1-C2 bond, C2-C3 bond, or C3-C4 bond. The 3-hydroxy-5-(3,3,5,7-tetrahydroxy-4-oxochroman-2-yl) cyclohexa-3,5-diene-1, 2-dione (m/z 333.0244) and unknown compound m/z 435.0925 were annotated as key intermediates in DMY degradation. Four phenolic acids, including 3,4,5-trihydroxybenzoic acid (m/z 169.0136, RT 1.4 min), 2,4,6-trihydroxyphenylglyoxylic acid (m/z 197.0084, RT 1.7 min), 2-oxo-2-(2,4,6-trihydroxyphenyl) acetaldehyde (m/z 181.0132, RT 2.4 min), and 2,4,6-trihydroxybenzoic acid (m/z 169.0139, RT 2.5 min) were identified as the major end products of DMY degradation. In addition, 5-((3,5dihydroxyphenoxy) methyl)-3-hydroxycyclohexa-3,5-diene-1,2-dione (m/z 261.0399, RT 11.7 min) and unidentified compound with m/z 329.0507 (RT 1.0 min) were also suggested to be end products of DMY degradation. These results provide novel insights on DMY stability and degradation products. Moreover, the heat treatment of DMY aqueous solution was found to gradually reduce the antioxidant activities of DMY, and even destroy the beneficial effect of DMY on the gut microbiota composition.

Evaluation of pharmacokinetic parameters, safety and tolerability of single and multiple doses of Vespireit®: results of phase I clinical trial.

Objective: to evaluate the pharmacokinetic parameters, safety and tolerability of Vespireit® (INN buspirone), prolonged-release tablets, 15 mg (JSC “Valenta Pharm”, Russia), which is being developed as a drug for the treatment of functional vertigo, in healthy volunteers with a single oral dose of 15 and 30 mg on an empty stomach or after meals and multiple doses in a daily dose of 15 mg.Material and methods. JSC “Valenta Pharm” conducted an open-label, three-stage, randomized, two-periods, cross-over comparative clinical trial of Vespireit®. At stages 1 and 2, healthy volunteers were screened and randomized into two equal groups (Group 1 and Group 2), as well as the distribution of participants in Group 3. Then, at stage 1, healthy volunteers (n=24) took Vespireit® once at a dose of 15 mg on an empty stomach or after meals, at stage 2, volunteers (n=24) took Vespireit® once at a dose of 30 mg on an empty stomach or after meals. Stage 3 was conducted in 18 volunteers as a non-randomized, non-comparative study with Vespireit® 15 mg on an empty stomach once daily for 5 days. During the study, blood samples were collected from each subject to determine the concentration of test compounds in blood plasma and subsequently calculate their pharmacokinetic parameters. Quantitative determination of buspirone and its two metabolites, 6-hydroxybuspirone (6OH-bus) and 1-(2-pyrimidinyl)-piperazine (1-PP), was performed by a validated high-performance liquid chromatography method with tandem mass spectrometric detection. During the study, tolerability and safety were also evaluated: adverse events, vital signs, laboratory parameters and electrocardiogram parameters were recorded.Results. When the study drug Vespireit®, prolonged-release tablets, was administered once at a dose of 15 mg to healthy volunteers after a meal, an increase in the relative bioavailability and relative degree of absorption of buspirone was observed compared with the fasting state, which was accompanied by a 1.5- and 2.5-fold increase in the AUC0–t and Cmax of buspirone, respectively. In addition, the Cmax of 6-OH-bus increased 1.4-fold and the Cmax of 1-PP increased 1.2-fold. Meal had no effect on the AUC0–t of 6-OH-bus and 1-PP. When the study drug was administered once at a dose of 30 mg after a meal, an increase in the relative bioavailability and relative degree of absorption of buspirone was observed compared with fasting, which was accompanied by an increase in the AUC0–t and Cmax by 1.5- and 2.1-fold, respectively. A 1.2-fold increase in the Cmax of 6-OH-bus was also observed. Food had no effect on the AUC0–t of 6-OH-bus and on the AUC0–t and Cmax of 1-PP. No accumulation of buspirone and its metabolites was observed with repeated dosing.Conclusion. In the study, the values of pharmacokinetic parameters of the new drug Vespireit® in the prolonged-release tablets dosage form were determined both with single administration on an empty stomach and after meals and with multiple administration. The differences and advantages over buspirone preparations in the form of immediate-release tablets are shown: higher relative bioavailability of buspirone with a reduction in interindividual differences in bioavailability and Cmax, more stable plasma concentration of buspirone with reduced peak values of the active substance and metabolite 1-PP, a 1.2-fold increase in AUC0–∞, a 2-fold increase in T1/2, and a 2-fold increase in the degree of retardation (MRT) for buspirone, a 1.4-fold increase in Cmax and MRT of 6-OH-bus, a decrease in AUC0–∞, AUC0–t, and T1/2 of metabolite 1-PP, and a decrease in the ratio of the concentration of 1-PP to the concentration of buspirone and to 6-OH-bus. A favorable safety profile of Vespireit® was demonstrated.

Biomonitoring of perfluoroalkyl and polyfluoroalkyl substances (PFAS) from the Survey of the Health of Wisconsin (SHOW) 2014-2016 and comparison with the National Health and Nutrition Examination Survey (NHANES).

Per- and polyfluoroalkyl substances (PFAS) are a growing class of manufactured chemical compounds found in a variety of consumer products. PFAS are ubiquitous in the environment and were found in many humans sampled in the United States (U.S.). Yet, significant gaps in understanding statewide levels of exposure to PFAS remain. The goals of this study are to establish a baseline of exposure at the state level by measuring PFAS serum levels among a representative sample of Wisconsin residents and compare to United States National Health and Nutrition Examination Survey (NHANES). The study sample included 605 adults (18+ years of age) selected from the 2014-2016 sample of the Survey of the Health of Wisconsin (SHOW).Thirty-eight PFAS serum concentrations were measured using high-pressure liquid chromatography coupled with tandem mass spectrometric detection (HPLC-MS/MS) and geometric means were presented. Weighted geometric mean serum values of eight PFAS analytes from SHOW were compared to U.S. national levels from the NHANES 2015-2016 sample (PFOS, PFOA, PFNA, PFHxS, PFHpS, PFDA, PFUnDA), and the 2017-2018 sample for Me-PFOSA, PFHPS using the Wilcoxon rank-sum test. PFOS, PFHxS, PFHpS, PFDA, PFNA, and PFOA were detected in over 96% of SHOW participants. In general, SHOW participants had lower serum levels across all PFAS when compared to NHANES. Serum levels increased with age and were higher among males and whites. Similar trends were seen in NHANES, except non-whites had higher PFAS levels at higher percentiles in NHANES. The present study conducts biomonitoring of 38 PFAS among representative sample of residents in the state of Wisconsin. Results suggest that while the majority of Wisconsin residents tested have detectable levels of PFAS in their blood serum, they may have a lower body burden of some PFAS compared to a nationally representative sample. Older adults, males, and whites may have a higher body burden of PFAS relative to other groups, both in Wisconsin and the wider United States.

A High-Throughput Absolute Quantification of Protein-Bound Tryptophan from Model and Crop Seeds.

This protocol describes a high-throughput absolute quantification protocol for the aromatic essential amino acid, tryptophan (Trp). This procedure consists of a milligram-scale alkaline hydrolysis followed by an absolute quantification step using a multiple reaction monitoring tandem mass spectrometric (LC-MS/MS) detection method. The approach facilitates the analysis of a few hundred samples per week by using a 96-well plate extraction setup. Importantly, the method uses only ∼4 mg of tissue per sample and uses the common alkaline hydrolysis protocol, followed by water extraction that includes L-Trp-d5 as an internal standard to enable the quantification of the absolute level of the bound Trp with high precision, accuracy, and reproducibility. The protocol described herein has been optimized for seed samples for Arabidopsis thaliana, Glycine max, and Zea mays but could be applied to other plant tissues. © 2023 Wiley Periodicals LLC. Basic Protocol: Analysis of protein-bound tryptophan from seeds.