Methods in food defence: a simple and robust screening for 16 rodenticides by LC-DAD/FLD following QuEChERS-based extraction.

Fortunately, the intentional contamination of food or water supplies out of criminal or terroristic motivation is a rather rare event. However, in the face of asymmetric warfare and as the consequences of such an event would be severe, food defence as a necessary supplement to food safety is gaining increased attention. While some progress has been made in developing non-target detection devices, the contamination of food or water supplies using readily available rodenticides may still be revealed only by complex analytical techniques. The presented study therefore aimed to develop a quick and easy screening method for the detection of sixteen globally common rodenticides in foodstuffs. Robust operation with limited personnel and analytical resources were one benchmark to be met by the method, which uses a slightly modified QuEChERS (quick, easy, cheap, effective, rugged, safe) protocol for dispersive solid-phase extraction and subsequent ion-pair chromatography with diode-array and fluorescence detection. Quantification limits were as low as 5 µg/kg with satisfying bias (recovery) and repeatability rates of 77 to 117% and 1.8 to 17.1%, respectively. The developed method provides reliable and robust detection of these deadly poisons at toxic concentrations, which was demonstrated impressively in an improvised assault scenario.

Aminoalkoxy-substituted coumarins: Synthesis and evaluation for reactivation of inhibited human acetylcholinesterase.

Reactivation of inhibited acetylcholinesterase remains an important therapeutic strategy for the treatment of poisoning by organophosphorus compounds, such as nerve agents or pesticides. Although drugs like obidoxime or pralidoxime have been used with considerable success, there is a need for new substances capable of reactivating acetylcholinesterase with a broader scope and increased efficacy. Possible screening candidates must fulfill two fundamental requirements: They must (i) show an affinity to acetylcholinesterase well balanced between sufficient binding and competitive inhibition and (ii) facilitate the nucleophilic cleavage of the phosphorylated catalytic serine residue. We attached a variety of nonaromatic primary and secondary amines to a coumarin core through selected alkoxy side linkers attached at coumarin positions 6 or 7 to obtain a small set of possible reactivators. Evaluation of their inhibition and reactivation potential in vitro showed some activity with respect to acetylcholinesterase inhibited by cyclosarin.

Guiding Molecularly Imprinted Polymer Design by Pharmacophore Modeling.

Molecularly imprinted polymers (MIP) combine the selectivity of immunoaffinity chromatography with the robustness of common solid-phase extraction in what is referred to as molecularly imprinted solid-phase extraction (MISPE). This contribution shows how MIP design may be guided by pharmacophore modeling for the example of citrinin, which is an emerging mycotoxin from cereals. The obtained pharmacophore model allowed searching public databases for a set of citrinin-mimicking molecular surrogates. Imprinted and non-imprinted polymers were subsequently obtained through bulk and core-shell polymerization in the presence of these surrogates. Evaluation of their binding ability for citrinin and structurally related ochratoxin A revealed a promising MIP derived from rhodizonic acid. A protocol for MISPE of citrinin from cereals was subsequently developed and compared to immunoaffinity chromatography with respect to clean-up efficiency and recovery.

Myco-DES: Enabling Remote Extraction of Mycotoxins for Robust and Reliable Quantification by Stable Isotope Dilution LC-MS/MS.

Mycotoxins remain a global threat to human and animal health, especially in countries lacking effective measures to detect and control contaminated commodities. As the quantification of mycotoxins usually relies on complex and expensive techniques, the availability of suitable instrumentation is often a bottleneck in reliable mycotoxin detection. As part of our research toward strategies offering widespread access to mycotoxin analysis while cutting down on costs, we present a new extraction and quantification protocol combining materials originally designed for dried blood spot analysis with stable isotope dilution analysis. Its key benefits are that extraction of mycotoxins can be carried out at remote sites and by minimally trained personnel, while quantification will take place in specialized central laboratories simply connected by regular, paper-based mail. As a proof of concept, aflatoxins, ochratoxin A, and deoxynivalenol were extracted from cereal-based foodstuffs, fixed on paper cards for transport, and successfully quantified after re-extraction by stable isotope dilution LC-MS/MS analysis. Several materials (cellulose/polyethylene terephthalate/glass fiber, nontreated/chemically treated) as well as possible transport and storage conditions (temperature, humidity) were evaluated. The final myco-DES (dried extract spots) protocol allows quantification of mycotoxin levels currently recognized as safe (aflatoxin B1: 2 µg/kg, ochratoxin A: 3 µg/kg, deoxynivalenol: 500 µg/kg) after a storage of up to 4 weeks under tropical climate conditions (40 °C, 75% relative humidity).

Bioanalytical verification of V-type nerve agent exposure: simultaneous detection of phosphonylated tyrosines and cysteine-containing disulfide-adducts derived from human albumin.

Nerve agents still represent a serious threat to civilian and military personnel as demonstrated by the violent conflict in the Middle East. For verification of poisoning, covalent adducts with endogenous proteins (e.g., human serum albumin, HSA) are valuable long-term biomarkers. Accordingly, we developed a microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS) method for simultaneous detection of HSA-adducts with the V-type nerve agents VX, Chinese VX (CVX), and Russian VX (RVX). Following Pronase-catalyzed proteolysis, novel disulfide-adducts were detected in addition to phosphonylated tyrosine residues. Dipeptide disulfide-adducts were formed between the thiol-containing leaving group of the V-type nerve agents (2-(diisopropylamino)ethanethiol, DPAET, for VX and 2-(diethylamino)ethanethiol, DEAET, for CVX and RVX) and the free thiol group of Cys34 in HSA (DPAET-CysPro, DEAET-CysPro). We also identified tripeptide disulfide-adducts containing Cys448 (MetProCys-DPAET, MetProCys-DEAET) and to a lesser extent Cys514 (AspIleCys-DPAET, AspIleCys-DEAET). Synthetic tripeptide references were used for confirmation of the postulated structures by µLC-ESI MS/HR MS. Lower limits of detection were determined in human plasma, being nearly identical for the three V-type nerve agents, and corresponded to 1-6 µM nerve agent for tyrosine-adducts, 1-3 µM nerve agent for CysPro-adducts, and 6 µM nerve agent for MetProCys-adducts, thus covering concentrations of toxicological relevance. Characterization of proteolysis kinetics revealed stable plateaus for all adducts being reached between 60 and 90 min at 37 °C. Adduct formation kinetics were characterized by simultaneously monitoring the V-type nerve agent, its leaving group, and the corresponding disulfide dimer. Furthermore, adduct formation patterns were investigated as a function of the molar ratio of HSA to V-type nerve agent. Graphical abstract Modification of human serum albumin (HSA) by V-type nerve agents Chinese VX (CVX) and RussianVX (RVX). Various tyrosine residues (Tyr???)n (e.g. most reactive Tyr411) were phosphonylated and disulfide-adducts were formed between the thiol-containing leaving group 2-(diethylamino)ethanethiol (DEAET) and at least three cysteine residues (Cys34, Cys448 and Cys514). Pronase-mediated proteolysis produced low-molecular cleavage products including phosphonylated tyrosines, dipeptide (Cys34Pro) and tripeptide (MetProCys448, AspIleCys514) disulfide-adducts that were detected by microbore liquid chromatography-electrospray ionization mass spectrometry/high-resolution mass spectrometry (µLC-ESI MS/HR MS).

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols.

The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (+)-taxifolin but not that of (-)-taxifolin. The Km for (+)-taxifolin was 6.4 ± 0.8 µM, and the Vmax was 108 ± 4 µmol min-1 (mg protein)-1 The enzyme also isomerized (+)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439-1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus IMPORTANCE: Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria.

Inhibition of human pyridoxal kinase by 2-acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI).

2-Acetyl-4-((1R,2S,3R)-1,2,3,4-tetrahydroxybutyl)imidazole (THI) is observed as a minor contaminant in caramel food colourings (E 150c). Feeding experiments with rodents have revealed a significant lymphopenic effect that has been linked to the presence of THI in these food colourings. Pyridoxal kinase inhibition by THI has been suggested, but not demonstrated, as a mode of action as it leads to lowered levels of pyridoxal-5'-phosphate, which are known to cause lymphopenia. Recently, THI was also shown to inhibit sphingosine-1-phosphate lyase causing comparable immunosuppressive effects and derivatives of THI are being developed for the treatment of rheumatoid arthritis in humans. Interestingly, sphingosine-1-phosphate lyase activity depends on pyridoxal-5'-phosphate, which in turn is provided by pyridoxal kinase. This report shows that THI does inhibit pyridoxal kinase with competitive and mixed-type non-competitive behaviour towards its two substrates, pyridoxal and ATP, respectively. The corresponding inhibition constants are in the low millimolar range.

The Chemotype of Chromanones as a Privileged Scaffold for Multineurotarget Anti-Alzheimer Agents.

The multifactorial nature of Alzheimer's disease necessitates the development of agents able to interfere with different relevant targets. A series of 22 tailored chromanones was conceptualized, synthesized, and subjected to biological evaluation. We identified one representative bearing a linker-connected azepane moiety (compound 19) with balanced pharmacological properties. Compound 19 exhibited inhibitory activities against human acetyl-, butyrylcholinesterase and monoamine oxidase-B, as well as high affinity to both the σ1 and σ2 receptors. Our study provides a framework for the development of further chromanone-based multineurotarget agents.

Development of a candidate reference method for the simultaneous quantitation of the boar taint compounds androstenone, 3α-androstenol, 3ß-androstenol, skatole, and indole in pig fat by means of stable isotope dilution analysis-headspace solid-phase microextraction-gas chromatography/mass spectrometry.

The steroidal pig pheromones androstenone (5α-androst-16-en-3-one), 3α-androstenol (5α-androst-16-en-3α-ol), and 3ß-androstenol (5α-androst-16-en-3ß-ol) as well as the heterocyclic aromatic amines skatole and indole, originating from microbial degradation of tryptophan in the intestine of pigs, are frequently recognized as the major compounds responsible for boar taint. A new procedure, applying stable isotope dilution analysis (SIDA) and headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) for the simultaneous quantitation of these boar taint compounds in pig fat was developed and validated. The deuterated compounds androstenone-d(3), 3ß-androstenol-d(3), skatole-d(3), and indole-d(6) were synthesized and successfully employed as internal standards for SIDA. The new procedure is characterized by a fast, simple, and economic sample preparation: methanolic extraction of the melted fat followed by a freezing and an evaporation step allows for extraction and enrichment of all five analytes. Additional time-consuming cleanup steps were not necessary, as HS-SPME sampling overcomes fat-associated injector and column contamination. The method has been validated by determining intra- and interday precision and accuracy as well as the limit of detection (LOD) and limit of quantitation (LOQ). Additionally, a cross-validation for androstenone, skatole, and indole was carried out comparing the results of 25 back fat samples obtained simultaneously by the new SIDA-HS-SPME-GC/MS procedure with those obtained in separate GC/MS and high-performance liquid chromatography fluorescence detection (HPLC-FD) measurements. The cross-validation revealed comparable results and confirms the feasibility of the new SIDA-HS-SPME-GC/MS procedure.

The thermal and enzymatic taxifolin-alphitonin rearrangement.

This report describes a detailed investigation of the thermal and enzymatic conversion of taxifolin to alphitonin. Chromatographic separation of the four dihydroquercetin stereoisomers 1-4 in combination with circular dichroism spectroscopy permitted elucidation of the kinetics of this rearrangement and characterization of the different reaction pathways involved. Our findings are corroborated by quantum chemistry calculations that reveal a unique cascade of tautomerization processes leading from taxifolin to alphitonin and also explain the racemization of alphitonin at room temperature. Furthermore, the substrate specificity toward (+)-taxifolin of an enzyme from Eubacterium ramulus catalyzing this intriguing rearrangement is demonstrated.

In vivo labelling of hippocampal beta-amyloid in triple-transgenic mice with a fluorescent acetylcholinesterase inhibitor released from nanoparticles.

The drastic loss of cholinergic projection neurons in the basal forebrain is a hallmark of Alzheimer's disease (AD), and drugs most frequently applied for the treatment of dementia include inhibitors of the acetylcholine-degrading enzyme acetylcholinesterase (AChE). This protein is known to act as a ligand of beta-amyloid (Abeta) in senile plaques, a further neuropathological sign of AD. Recently, we have shown that the fluorescent, heterodimeric AChE inhibitor PE154 allows for the histochemical staining of cortical Abeta plaques in triple-transgenic (TTG) mice with age-dependent beta-amyloidosis and tau hyperphosphorylation, an established animal model for aspects of AD. In the present study, we have primarily demonstrated the targeting of Abeta-immunopositive plaques with PE154 in vivo for 4 h up to 1 week after injection into the hippocampi of 13-20-month-old TTG mice. Numerous plaques, double-stained for PE154 and Abeta-immunoreactivity, were revealed by confocal laser-scanning microscopy. Additionally, PE154 targeted hippocampal Abeta deposits in aged TTG mice after injection of carboxylated polyglycidylmethacrylate nanoparticles delivering the fluorescent marker in vivo. Furthermore, biodegradable core-shell polystyrene/polybutylcyanoacrylate nanoparticles were found to be suitable, alternative vehicles for PE154 as a useful in vivo label of Abeta. Moreover, we were able to demonstrate that PE154 targeted Abeta, but neither phospho-tau nor reactive astrocytes surrounding the plaques. In conclusion, nanoparticles appear as versatile carriers of AChE inhibitors and other promising drugs for the treatment of AD.

A gorge-spanning, high-affinity cholinesterase inhibitor to explore beta-amyloid plaques.

Cholinesterases are involved in the pathological formation of beta-amyloid plaques. To investigate this pathohistological hallmark of Alzheimer's disease we prepared a high-affinity, fluorescent cholinesterase inhibitor. Its fluorescence intensity was significantly enhanced upon binding to cholinesterases. Using this probe, brain samples from mice and humans affected by Alzheimer's disease were successfully analyzed for beta-amyloid plaques. Unexpectedly, it was discovered, by competition experiments, that the compound binds to amyloid structures, rather than to cholinesterases inside of the plaques.

Determination of the Synthetic Antioxidant Ethoxyquin and Its Metabolites in Fish and Fishery Products Using Liquid Chromatography-Fluorescence Detection and Stable-Isotope Dilution Analysis-Liquid Chromatography-Tandem Mass Spectrometry.

The use of the synthetic antioxidant ethoxyquin (1,2-dihydro-6-ethoxy-2,2,4-trimethylquinoline, EQ) as a flame retardant in fish meal transported by sea is required by international authorities to prevent self-ignition. Because of extensive carry-over within the food chain, selective and sensitive analytical methods are required for investigations on human exposure and the safety of EQ and its metabolites. Therefore, a simple, fast, and rugged liquid-chromatography (LC) method was developed for the detection of EQ and its metabolites in fish and fishery products after liquid-liquid extraction using QuEChERS. For screening purposes, a fluorescence detector was used (LC-FLD) with the EQ-analogue methoxyquin serving as an internal standard. For stable-isotope dilution analysis by liquid chromatography-tandem mass spectrometry (SIDA-LC-MS/MS), deuterated analogues of EQ and its metabolites were synthesized for the first time and allowed for sensitive quantification and thus confirmation of screening results. Both methods were validated and successfully applied to commercially available fish samples.

Optimized Analysis of Ergot Alkaloids in Rye Products by Liquid Chromatography-Fluorescence Detection Applying Lysergic Acid Diethylamide as an Internal Standard.

Analysis of ergot alkaloids remains a topic of importance and the European Food Safety Authority (EFSA) has encouraged laboratories to provide monitoring data for the further evaluation of their occurrence in food and feed. While LC-MS/MS has dominated developments in recent years, LC-FLD is still more widespread, especially in developing countries. To improve the analysis of ergot alkaloids by LC-FLD, we developed an improved protocol introducing lysergic acid diethylamide (LSD) for internal standardization. Several aspects such as the composition and pH of the extraction medium, type of sorbent and conditions applied for solid-phase extraction/clean-up, use of a keeper during final evaporation and the type of syringe filter used for filtration prior to injection were thoroughly investigated. Optimized conditions comprise extraction by ethyl acetate, methanol and 28% aqueous ammonia in combination with basic aluminum oxide for extract clean-up. Use of a keeper was found inappropriate as LC-FLD analysis was significantly affected by co-eluting keeper components. Similar observations were made with some of the investigated syringe filters, where polytetrafluoroethylene (PTFE) proved to be the most suitable. Validation and application of the optimized methodology to real samples provided limits of detection and quantification suitable for the evaluation of relevant ergot alkaloid contaminations in rye and bakery products with superior precision that was facilitated by the introduced internal standard, LSD.

Chromenones as Multineurotargeting Inhibitors of Human Enzymes.

The complex nature of multifactorial diseases, such as Morbus Alzheimer, has produced a strong need to design multitarget-directed ligands to address the involved complementary pathways. We performed a purposive structural modification of a tetratarget small-molecule, that is contilisant, and generated a combinatorial library of 28 substituted chromen-4-ones. The compounds comprise a basic moiety which is linker-connected to the 6-position of the heterocyclic chromenone core. The syntheses were accomplished by Mitsunobu- or Williamson-type ether formations. The resulting library members were evaluated at a panel of seven human enzymes, all of which being involved in the pathophysiology of neurodegeneration. A concomitant inhibition of human acetylcholinesterase and human monoamine oxidase B, with IC50 values of 5.58 and 7.20 µM, respectively, was achieved with the dual-target 6-(4-(piperidin-1-yl)butoxy)-4H-chromen-4-one (7).

First gallamine-tacrine hybrid: design and characterization at cholinesterases and the M2 muscarinic receptor.

Gallamine and tacrine are allosteric antagonists at muscarinic M2 acetylcholine receptors and inhibitors of acetylcholinesterase. At both acetylcholine-binding proteins, gallamine and tacrine are known to occupy two different binding sites: in M2 receptors within the allosteric binding area and in acetylcholinesterase at its catalytic and its peripheral site. To find new ligands of both targets, we designed a gallamine-tacrine dimer and several derived hybrid compounds to address the two binding sites. Their M2 receptor allosteric and acetylcholinesterase inhibitory potential was determined. The hybrid compounds revealed an allosteric potency in the low nanomolar range exceeding the allosteric potency of gallamine and tacrine by factors of 100 and 4800, respectively. Cholinesterase inhibition was augmented by hybrid formation, and all compounds exhibited IC50 values in the lower nanomolar range. Thus, gallamine-tacrine hybrid formation is a valuable approach toward high affinity ligands concurrently targeting these acetylcholine-binding proteins.

Identification of novel disulfide adducts between the thiol containing leaving group of the nerve agent VX and cysteine containing tripeptides derived from human serum albumin.

Chemical warfare agents represent a continuous and considerable threat to military personnel and the civilian population. Such compounds are prohibited by the Chemical Weapons Convention, to which adherence by the member states is strictly controlled. Therefore, reliable analytical methods for verification of an alleged use of banned substances are required. Accordingly, current research focuses on long-term biomarkers derived from covalent adducts with biomolecules such as proteins. Recently, we have introduced a microbore liquid chromatography/electrospray ionization high-resolution tandem mass spectrometry method allowing for the investigation of two different classes of adducts of the nerve agent VX with human serum albumin (HSA). Phosphonylated tyrosine residues and novel disulfide adducts at cysteine residues of HSA were produced by enzymatic cleavage with pronase and detected simultaneously. Notably, the thiol containing leaving group of VX (2-(diisopropylamino)ethanethiol, DPAET) formed disulfide adducts that were released as cysteine and proline containing dipeptides originating from at least two different sites of HSA. Aim of this study was to identify assumed and novel adducts of DPAET with HSA using synthetic peptide reference compounds. Two novel tripeptides were identified representing disulfide adducts with DPAET (Met-Pro-Cys-DPAET, MPC-DPAET and Asp-Ile-Cys-DPAET, DIC-DPAET). MPC-DPAET was shown to undergo partial in-source decay during electrospray ionization for MS detection thereby losing the N-terminal Met residue. This results in the more stable Pro-Cys-DPAET (PC-DPAET) dipeptide detectable as protonated ion. The limit of detection for MPC-DPAET was evaluated, revealing toxicologically relevant VX plasma concentrations. The results provide novel insights into the reactivity of VX and its endogenous targets. Copyright © 2016 John Wiley & Sons, Ltd.

Novel heterobivalent tacrine derivatives as cholinesterase inhibitors with notable selectivity toward butyrylcholinesterase.

Two series of novel heterobivalent tacrine derivatives were synthesized. A trimethoxy substituted benzene was linked to the tacrine moiety by a hydrazide-based linker. The compounds were evaluated as cholinesterase inhibitors, and trimethoxybenzoic acid derivatives with 11- or 12-atom spacers were the most potent inhibitors of human acetylcholinesterase. The inhibitors showed a surprising selectivity toward human butyrylcholinesterase, where several trimethoxyphenylpropionic acid derivatives had IC(50) values less than 250 pM.

2-Aminoacetophenone Is the Main Volatile Phase I Skatole Metabolite in Pietrain × Baden-Württemberg Hybrid Type Boars.

Skatole metabolites have been considered as putative contributors to boar taint. Recently, 2-aminoacetophenone, a volatile phase I skatole metabolite, was identified in back fat samples from boars of Pietrain × Baden-Württemberg hybrid type. This paper addresses the question of the physiological origin of the observed 2-aminoacetophenone in these pigs. Microsomal fractions from nine boars were isolated, and formation of skatole metabolites was subsequently analyzed by stable-isotope dilution analysis (SIDA) using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME/GC-MS). Significant breed-related differences in phase I skatole metabolism were observed, explaining the high levels of 2-aminoacetophenone in Pietrain × Baden-Württemberg hybrid type boars.

A novel fluorogenic probe for the investigation of free thiols: Application to kinetic measurements of acetylcholinesterase activity.

A novel coumarin-derived thiol probe, based on the thiol-promoted cleavage of a quenching 2,4-dinitrobenzenesulfonyl group is described. The probe shows a sensitive fluorescence turn-on and sufficient solubility in aqueous environments. As a proof of concept, a new assay for AChE activity was developed as a useful addition to the established Ellman method. The observed reaction kinetics followed an asymmetric sigmoidal pattern and were successfully evaluated applying a three parameter Gompertz equation. Providing a linear relationship between the detected fluorescence formation curves and corresponding enzyme activities, this probe appears as a valuable tool for AChE activity measurements.