False Results Caused by Solvent Impurity in Tetrahydrofuran for MALDI TOF MS Analysis of Amines

Tetrahydrofuran (THF) is one of the most frequently used solvents in the MALDI TOF MS analysis of synthetic compounds. However, it should be used with caution because a trace amount of 4-hydroxybutanal (HBA) might be generated and accumulated in THF during storage. Since only a tiny amount of analytes is required in MALDI MS measurements, a trace amount of HBA might have a significant effect on the MS results. It was found that HBA will quickly react with primary and secondary amino compounds, leading to false results about the sample composition with an extra series of ions with additional mass of 70 Da in between. The formation of HBA can be inhibited by butylated hydroxytoluene (BHT) antioxidant. Therefore, when THF is required as the solvent for sample preparation, it is strongly recommended to use a BHT-stabilized one, at least for the analysis of compounds with amino groups.

In vivo 1H-NMR procedure to determine several rat cerebral metabolite levels simultaneously, undisturbed by water and lipid signals

Methods developed for in vivo 1H-NMR spectroscopy are evaluated and applied using conscious rats. Good quality 1H-spectra of the brain are obtained using a surface coil and a spin echo pulse sequence with the binomial 1-1 and 2-2 water suppression pulses. However, comparing spectra from various rats with each other the water and lipid signals, which cause spectral overlap problems, may differ while the other spectral peaks agree well. Spatially one- and two-dimensional 1H spectroscopic imaging of the rat brain shows that the former signals stem from distinct spatial regions localized close to the rf coil. From a spectroscopic image, a spectrum over a limited spatial region is constructed in which the water signals are strongly reduced, the lipid signals are eliminated and lactic acid can be observed clearly simultaneously with other metabolites.     

The German bakery waste incident; use of a combined approach of screening and confirmation for dioxins in feed and food

During the last six years several incidents have occurred with dioxins in feed, stressing the need for rapid screening methods for these compounds. The most recent incident was the contamination of bakery waste used for animal feed due to the use of waste wood for drying of the material. In addition to Germany, the material was also shipped to the Netherlands. Levels up to 12 ng TEQ/kg have been detected, being about 15 times over the current limit of 0.75 ng TEQ/kg. In the Netherlands a combined strategy of screening with the CALUX-bioassay and the HRGC/HRMS confirmatory method was used to rapidly control the incident. Pigs were contaminated by the incident but only to a very limited extent. Despite the rather low limits for pig meat, the CALUX bioassay showed excellent performance, once again confirming the value of this assay.     

Bioactivity-based screening of antibiotics and hormones

Bioactivity-based screening methods are relatively cheap, quick and easy to use tools. Especially with respect to antimicrobial residues and compounds with hormonal activity, they form a very cost-effective alternative to physical chemical methods in large-scale surveillance and monitoring programs, where their main purpose is to identify samples that require additional chemical confirmation. A major advantage is their intrinsic capability to detect unknown compounds and new hazards. This review shows an overview of the available methods and their potential and limitations for regulatory control.     

Influence of the Solvent and the Enantiomeric Purity on the Transition between Different Supramolecular Polymers

The self‐assembly of two enantiomerically pure hexa(oligo (p‐phenylene vinylene))‐substituted benzenes having 24 stereocenters was studied in pure methylcyclohexane (MCH) and in a mixture of MCH/toluene (4:1). Irrespective of the solvent a cooperative supramolecular polymerization mechanism was determined for these star‐shaped molecules by using temperature‐dependent CD and UV/Vis spectroscopy. Quite remarkably, a transition from one helical supramolecular state (A) to a second more thermodynamically stable supramolecular helical assembly (B) was observed. The rate of the A→B transition was strongly dependent on the nature of the solvent; being faster in the solvent mixture than in pure MCH. By using size exclusion chromatography we could relate the increased rate to a decreased stability of the supramolecular A state in the solvent mixture. Next, we mixed the two enantiomerically pure hexa‐substituted benzene derivatives in a so‐called majority‐rules experiment, which lead to the anitcipated chiral amplification in the A state. More importantly it appeared that the A→B transition was significantly hampered in these mixed systems. Furthermore, the absence of chiral amplification in the B state revealed the formation of separated enantiomerically pure assemblies. Therefore, by using a wide variety of spectroscopic and chromatographic techniques we determined the influence of solvent and enantiomeric purity on the transition between different supramolecular states.     

Bioassays for the detection of growth-promoting agents, veterinary drugs and environmental contaminants in food

Residues of growth-promoting agents, veterinary drugs and environmental contaminants in food products are routinely analyzed with chemical-analytical methods, using physical and spectrometric properties of a compound. Since residue limits are in general based on biological properties of compounds, bioassays offer in theory a good alternative. As a consequence, these assays are much more suitable for the detection of mixtures of compounds with common biological properties, including possibly unknown agonists. Using modern molecular biological techniques, a new generation of bioassays has been developed, showing in general a higher sensitivity and specificity for the target compounds. The CALUX (chemical activated luciferase expression) assay was developed for the detection of polyhalogenated compounds, based on their affinity for the aryl hydrocarbon (Ah) receptor. This paper focuses on the specificity of the assay. The benzimidazole compounds oxfendazole, fenbendazole, febantel, thiabendazole, mebendazole, omeprazole, lanzoprazole and benomyl were shown to give a positive response in the assay. Similar results were obtained with dexamethasone, corticosterone and cortisol, which in addition were able to enhance the response obtained with TCDD. Similarly to the flavonoids alpha- and beta-naphtoflavone, the reported Ah receptor antagonist 4-amino-3-methoxyflavone showed a strong positive response at a concentration of 400 microM, but failed to inhibit the response obtained with TCDD. It is concluded that the chances of false-negative results appear to be minimal and can be recognized. False-positive or, better, unwanted results are in theory more likely to occur. Possible solutions to avoid or detect these type of results are discussed. In general, these kinds of assays offer great possibilities for screening of food samples. In addition to the further optimization of these assays, future work should be focused on the development of rapid, sample and selective extraction procedures.     

Bioactivity screening and mass spectrometric confirmation for the detection of PPARδ agonists that increase type 1 muscle fibres

Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARδ). Exposure of U937 cells to the PPARδ agonist GW501516 resulted in a marked increase in mRNA expression of the PPARδ target gene Angptl4 which was quantified by qRT-PCR analysis. Exposure of HepG2 cells transiently transfected with a PPARδ expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARδ agonists GW501516, GW610742 and L-165041 resulted in clear dose–response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2-based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46 V) and 454.07>257.08 (CE 30 V); for GW610742, 472.07>206.2 (CE 48 V) and 472.07>275.08 (CE 30 V); and for L-165041, 401.2>193.15 (CE 26 V) and 401.2>343.2 (CE 20 V).     

Evidence of the indirect hormonal activity of prohormones using liver S9 metabolic bioactivation and an androgen bioassay

Prohormones such as dehydroepiandrosterone (DHEA) are steroid precursors that do not show hormonal activity by themselves. Abuse of these prohormones in cattle fattening is hard to prove because of strong in vivo metabolism and the difficulty to detect metabolites which are not significantly above endogenous levels. The aim of the present work was to develop an in vitro assay capable of detecting the indirect hormonal activity of prohormones that might be present in feed supplements and injection preparations. Sample extracts were incubated with a bovine liver S9 fraction in order to mimic the in vivo metabolic activation. Subsequently incubated extracts were exposed to a highly androgen-specific yeast bioassay to detect hormonal activity. Metabolic activation of DHEA, 4-androstene-3,17-dione (4-adione) and 5-androstene-3,17-diol (5-adiol) resulted in an increased androgenic activity caused by the formation of the active androgen 17β-testosterone (17β-T), as shown by ultra-performance liquid chromatography and time-of-flight mass spectrometry with accurate mass measurement. The developed in vitro system successfully mimics the hydroxysteroid dehydrogenase (HSD)- and cytochrome P450-mediated in vivo metabolic transitions, thus allowing assessment of both bioactivity and chemical identification without the use of animal experiments. Screening of unknown supplement samples claimed to contain DHEA resulted in successful bioactivation and positive screening results according to the androgen yeast biosensor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An Inexpensive and Recyclable Silver‐Foil Catalyst for the Cyclopropanation of Alkenes with Diazoacetates under Mechanochemical Conditions

The diastereoselective cyclopropanation of various alkenes with diazoacetate derivatives can be achieved under mechanochemical conditions using metallic silver foil and a stainless‐steel vial and ball system. This solvent‐free method displays analogous reactivity and selectivity to solution‐phase reactions without the need for slow diazoacetate addition or an inert atmosphere. The heterogeneous silver‐foil catalyst system is easily recyclable without any appreciable loss of activity or selectivity being observed. The cyclopropanation products were obtained with excellent diastereoselectivities (up to 98:2 d.r.) and in high yields (up to 96 %).