Florian Hübner

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies

This paper reports theoretical and experimental studies of gas-phase fragmentation reactions of four naturally occurring isoflavones. The samples were analyzed in negative ion mode by direct infusion in ESI-QqQ, ESI-QqTOF and ESI-Orbitrap systems. The MS/MS and MS(n) spectra are in agreement with the fragmentation proposals and high-resolution analyses have confirmed the formulae for each ion observed. As expected, compounds with methoxyl aromatic substitution have showed a radical elimination of •CH(3) as the main fragmentation pathway. A second radical loss (•H) occurs as previously observed for compounds which exhibit a previous homolytic •CH(3) cleavage (radical anion) and involves radical resonance to stabilize the anion formed. However, in this study we suggest another mechanism for the formation of the main ions, on the basis of the enthalpies for each species. Compounds without methoxy substituent dissociate at the highest energies and exhibit the deprotonated molecule as the most intense ion. Finally, energy-resolved experiments were carried out to give more details about the gas-phase dissociation reaction of the isoflavones and the results are in agreement with the theoretical approaches.

Intestinal metabolism of two A-type procyanidins using the pig cecum model: detailed structure elucidation of unknown catabolites with Fourier transform mass spectrometry (FTMS).

Procyanidins, as important secondary plant metabolites in fruits, berries, and beverages such as cacao and tea, are supposed to have positive health impacts, although their bioavailability is yet not clear. One important aspect for bioavailability is intestinal metabolism. The investigation of the microbial catabolism of A-type procyanidins is of great importance due to their more complex structure in comparison to B-type procyanidins. A-type procyanidins exhibit an additional ether linkage between the flavan-3-ol monomers. In this study two A-type procyanidins, procyanidin A2 and cinnamtannin B1, were incubated in the pig cecum model to mimic the degradation caused by the microbiota. Both A-type procyanidins were degraded by the microbiota. Procyanidin A2 as a dimer was degraded by about 80% and cinnamtannin B1 as a trimer by about 40% within 8 h of incubation. Hydroxylated phenolic compounds were quantified as degradation products. In addition, two yet unknown catabolites were identified, and the structures were elucidated by Fourier transform mass spectrometry.

Identification and apoptotic potential of T-2 toxin metabolites in human cells

The mycotoxin T-2 toxin, produced by various Fusarium species, is a widespread contaminant of grain and grain products. Knowledge about its toxicity and metabolism in the human body is crucial for any risk assessment as T-2 toxin can be detected in processed and unprocessed food samples. Cell culture studies using cells of human origin represent a potent model system to study the metabolic fate of T-2 toxin as well as the cytotoxicity in vitro. In this study the metabolism of T-2 toxin was analyzed in a cell line derived from human colon carcinoma cells (HT-29) and primary human renal proximal tubule epithelial cells (RPTEC) using high-performance liquid chromatography coupled with Fourier transformation mass spectrometry (HPLC-FTMS). Both cell types metabolized T-2 toxin to a variety of compounds. Furthermore, cell cycle analysis in RPTEC proved the apoptotic effect of T-2 toxin and its metabolites HT-2 toxin and neosolaniol in micromolar concentrations.

Stable Isotope Dilution Analysis of Small Molecules with Carboxylic Acid Functions Using 18O Labeling for HPLC-ESI-MS/MS: Analysis of Fumonisin B1

(18)O labeling is a well-known method for the stable isotope labeling of proteins and peptides. This study describes a modified procedure for using (18)O labeling on small molecules. Fumonisin B1, a worldwide occurring mycotoxin, which is routinely analyzed by HPLC-MS/MS, was chosen as model compound. (18)O labeling was achieved by acid-catalyzed oxygen exchange from H2(18)O. A mixture of different isotopologues was obtained from the exchange, which, however, could be used as an internal standard for HPLC-MS/MS analysis. The identity of the (18)O-labeled fumonisin B1 was confirmed by NMR and HRMS measurements. The applicability as internal standard has been verified by comparison of results obtained from the method described in this paper to results obtained by reference methods. The presented method is of special interest as the (18)O labeling can be generally applied to a large group of small molecules containing carboxylic groups.

Systematic approach for structure elucidation of polyphenolic compounds using a bottom-up approach combining ion trap experiments and accurate mass measurements.

Polyphenols are a group of plant secondary metabolites with a wide range of structural differences. In many cases, in vitro and in vivo studies of polyphenols revealed beneficial health effects. The mass spectrometric characterization of polyphenols can be the key to understanding the metabolism and resorption of this group of substances. For structure elucidation of polyphenolic compounds nuclear magnectic resonance spectroscopy is the method of choice. Due to the broad structure variability and the sometimes relatively low concentrations of polyphenols and/or their metabolites in foods as well as physiological samples, mass spectrometry could be an alternative for structure elucidation. Especially high-resolution mass spectrometry, for example, Fourier transformation mass spectrometry (FTMS), is a valuable tool. Using a FTMS system, a systematic approach to the fragmentation behavior of phenolic and polyphenolic compounds was chosen to verify the influence of the structure on the fragmentation pattern of the different substances. Depending on the structure, specific fragment ions could be detected. Therefore, it is possible to gain reliable information about the structure of the pseudomolecular ion from its fragmentation spectrum, which is of great aid in the structure elucidation of unknown polyphenols and/or their metabolites.

Toxicity of arsenite and thio-DMAV after long-term (21 days) incubation of human urothelial cells: cytotoxicity, genotoxicity and epigenetics

This study aims to further mechanistically understand toxic modes of action after chronic inorganic arsenic exposure. Therefore long-term incubation studies in cultured cells were carried out, to display chronically attained changes, which cannot be observed in the generally applied in vitro short-term incubation studies. Particularly, the cytotoxic, genotoxic and epigenetic effects of an up to 21 days incubation of human urothelial (UROtsa) cells with pico- to nanomolar concentrations of iAsIII and its metabolite thio-DMAV were compared. After 21 days of incubation, cytotoxic effects were strongly enhanced in the case of iAsIII and might partly be due to glutathione depletion and genotoxic effects on the chromosomal level. These results are in strong contrast to cells exposed to thio-DMAV. Thus, cells seemed to be able to adapt to this arsenical, as indicated among others by an increase in the cellular glutathione level. Most interestingly, picomolar concentrations of both iAsIII and thio-DMAV caused global DNA hypomethylation in UROtsa cells, which was quantified in parallel by 5-medC immunostaining and a newly established, reliable, high resolution mass spectrometry (HRMS)-based test system. This is the first time that epigenetic effects are reported for thio-DMAV; iAsIII induced epigenetic effects occur in at least 8000 fold lower concentrations as reported in vitro before. The fact that both arsenicals cause DNA hypomethylation at really low, exposure-relevant concentrations in human urothelial cells suggests that this epigenetic effect might contribute to inorganic arsenic induced carcinogenicity, which for sure has to be further investigated in future studies.

Short‐term biomarkers of apple consumption

Scope: Urinary biomarkers are used to estimate the nutritional intake of humans. The aim of this study was to distinguish between low, medium, and high apple consumption by quantifying possible intake biomarkers in urine samples after apple consumption by HPLC‐MS/MS. Apples were chosen as they are the most consumed fruits in Germany. Methods and Results: Thirty subjects took part in 7‐day study. They abstained from apples and apple products except for one weighed apple portion resembling one, two, or four apples. Before apple consumption and during the following days spot urine samples were collected. These urine samples were incubated with β‐glucuronidase, diluted, and directly measured by HPLC‐MS/MS. Phloretin, epicatechin, procyanidin B2, and quercetin were detected in urine using Scheduled MRMTM mode. Phloretin was confirmed as a urinary biomarker of apple intake and had the ability to discriminate between low or medium (one or two apples) and high apple consumption (four apples). The groups also differ in the excretion of epicatechin and procyanidin B2. Conclusion: Apple consumption can be monitored by urinary biomarkers for a period of at least 12 h after consumption. Furthermore the amount of apples consumed can be estimated by the concentration of certain biomarkers.

A Fungal N-Dimethylallyltryptophan Metabolite from Fusarium fujikuroi

The range of secondary metabolites (SMs) produced by the rice pathogen Fusarium fujikuroi is quite broad. Several polyketides, nonribosomal peptides and terpenes have been identified. However, no products of dimethylallyltryptophan synthases (DMATSs) have been elucidated, although two putative DMATS genes are present in the F. fujikuroi genome. In this study, the in vivo product derived from one of the DMATSs (DMATS1, FFUJ_09179) was identified with the help of the software MZmine 2. Detailed structure elucidation showed that this metabolite is a reversely N‐prenylated tryptophan with a rare form of prenylation. Further identified products probably resulted from side reactions of DMATS1. The genes adjacent to DMATS1 were analyzed; this showed no influence on the biosynthesis of the product.

Pharmacokinetics of the Experimental Non-Nucleosidic DNA Methyl Transferase Inhibitor N-Phthalyl-L-Tryptophan (RG 108) in Rats.

DNA methyl transferase (DNMT) inhibitors can re‐establish the expression of tumour suppressor genes in malignant diseases, but might also be useful in other diseases. Inhibitors in clinical use are nucleosidic cytotoxic agents that need to be integrated into the DNA of dividing cells. Here, we assessed the in vivo kinetics of a non‐nucleosidic inhibitor that is potentially free of cytotoxic effects and does not require cell division. The non‐specific DNMT inhibitor N‐phthalyl‐l‐tryptophan (RG 108) was injected subcutaneously in rats. Blood was drawn 0, 0.5, 1, 2, 4, 6, 8 and 24 hr after injection and RG 108 in plasma was measured by high‐performance liquid chromatography coupled to mass spectrometry. Trough levels and area under the curve (AUC) were significantly higher with multiple‐dose administration and cytochrome inhibition. In this group, time to maximal plasma concentration (tmax, mean ± S.D.) was 37.5 ± 15 min., terminal plasma half‐life was approximately 3.7 h (60% CI: 2.1–15.6 h), maximal plasma concentration (Cmax) was 61.3 ± 7.6 μM, and AUC was 200 ± 54 μmol·h/l. RG 108 peak levels were not influenced by cytochrome inhibition or multiple‐dose administration regimens. Maximal tissue levels (Cmax in μmol/kg) were 6.9 ± 6.7, 1.6 ± 0.4 and 3.4 ± 1.1 in liver, skeletal and heart muscle, respectively. We conclude that despite its high lipophilicity, RG 108 can be used for in vivo experiments, appears safe and yields plasma and tissue levels in the range of the described 50% inhibitory concentration of around 1 to 5 μM. RG 108 can therefore be a useful tool for in vivo DNMT inhibition.

Electrochemical simulation of metabolic reactions of the secondary fungal metabolites alternariol and alternariol methyl ether

AbstractMycotoxins are secondary plant metabolites that have been found to cause severe diseases in humans and livestock. Exposure can take place on a daily basis since mycotoxins can be found not only in food, animal food, and dietary supplements but also in materials used in buildings. For this work, the Alternaria toxins alternariol (AOH) and alternariol methyl ether (AME) are chosen as representatives for this relevant compound class and are investigated regarding their oxidative phase I metabolism using a combination of electrochemical (EC) oxidation and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). This previously established method has been proven to be a valuable tool for the electrochemical simulation of certain phase I metabolic reactions. A comparison of the electrochemically generated products with those formed during microsomal incubation demonstrates the potential of the method for the successful prediction of the main phase I metabolic reactions of mycotoxins. It can thus find use as a supportive method in the elucidation of the metabolic pathways of various mycotoxins. Graphical AbstractOn-line set-up used for the electrochemical simulation of metabolism reactions of Alternaria toxins. (a) Electrochemical oxidation is coupled directly to the ESI interface of the MS for the generation of mass voltammograms. (b) Implementation of an HPLC system allows a chromatographic separation of previously generated oxidation products.