Preparative isolation of huperzines A and B from Huperzia serrata by displacement centrifugal partition chromatography

The pH-zone refining centrifugal partition chromatography technique was used to separate the two acetylcholinesterase inhibitors huperzines A and B from a crude alkaloid extract of the club moss Huperzia serrata. Complete co-elution of huperzines A and B was initially observed with the well-known methyl tert-butyl ether-acetonitrile-water (4:1:5, v/v/v) solvent system with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer. An efficient biphasic system was designed on the basis of solvent association that provided selectivity in the elution mode: n-heptane/ethyl acetate/n-propanol/water (5:15:35:45, v/v/v/v). Lowering the bridge solvent content (n-propanol) of this system increased the polarity difference between the two phases thus adapting it to the pH-zone refining mode. Thus, the purification of these compounds was achieved using the biphasic system n-heptane/ethyl acetate/n-propanol/water (10:30:15:45, v/v/v/v) with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer.     

Single-laboratory validation of a multiplex flow cytometric immunoassay for the simultaneous detection of coccidiostats in eggs and feed

Coccidiostats are authorized in the European Union (EU) to be used as poultry feed additives. Maximum (residue) levels (M(R)Ls) have been set within the EU for consumer and animal protection against unintended carry-over, and monitoring is compulsory. This paper describes the single-laboratory validation of a previously developed multiplex flow cytometric immunoassay (FCIA) as screening method for coccidiostats in eggs and feed and provides and compares different approaches for the calculation of the cut-off levels which are not described in detail within Commission Decision 2002/657/EC. Comparable results were obtained between the statistical (reference) approach and the rapid approaches. With the most rapid approach, the cut-off levels for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (DNC) and monensin in egg, calculated as percentages of inhibition (%B/B0), were 60, 32, 76, 80 and 84, respectively. In feed, the cut-off levels for narasin/salinomycin, lasalocid, nicarbazin (DNC) and monensin were 70, 64, 72 and 78, respectively, and could not be determined for diclazuril. For all analytes, except for diclazuril in feed, the rate of false positives (false non-compliant) in blank samples was lower than 1 %, and the rate of false negatives (false compliant) at the M(R)Ls was below 5 %. Additionally, very good correlations (r ranging from 0.994 to 0.9994) were observed between two different analysers, a sophisticated flow cytometer (FlexMAP 3D^®) and a more cost-efficient and transportable planar imaging detector (MAGPIX^®), hence demonstrating adequate transferability.     

Solvent effect on the excited-state dynamics of analogues of the photoactive yellow protein chromophore

We previously reported that two analogues of the Photoactive Yellow Protein chromophore, trans-p-hydroxycinnamic acid (pCA(2-)) and its amide derivative (pCM-) in their deprotonated forms, undergo a trans-cis photoisomerization whereas the thioester derivative, trans-p-hydroxythiophenyl cinnamate (pCT-), does not. pCT- is also the only one to exhibit a short-lived intermediate on its excited-state deactivation pathway. We here further stress the existence of two different relaxation mechanisms for these molecules and examine the reaction coordinates involved. We looked at the effect of the solvent properties (viscosity, polarity, solvation dynamics) on their excited-state relaxation dynamics, probed by ultrafast transient absorption spectroscopy. Sensitivity to the solvent properties is found to be larger for pCT- than for pCA(2-) and pCM-. This difference is considered to reveal that either the relaxation pathway or the reaction coordinate is different for these two classes of analogues. It is also found to be correlated to the electron donor-acceptor character of the molecule. We attribute the excited-state deactivation of analogues bearing a weaker acceptor group, pCA(2-) and pCM-, to a stilbene-like photoisomerization mechanism with the concerted rotation of the ethylenic bond and one adjacent single bond. For pCT-, which contains a stronger acceptor group, we consider a photoisomerization mechanism mainly involving the single torsion of the ethylenic bond. The excited-state deactivation of pCT- would lead to the formation of a ground-state intermediate at the "perp" geometry, which would return to the initial trans conformation without net isomerization.     

Surface immobilization and mechanical properties of catanionic hollow faceted polyhedrons

We report here for the first time on surface immobilization of hollow faceted polyhedrons formed from catanionic surfactant mixtures. We find that electrostatic interaction with the substrate dominates their adhesion behavior. Using polyelectrolyte coated surfaces with tailored charge densities, polyhedrons can thus be immobilized without complete spreading, which allows for further study of their mechanical properties using AFM force measurements. The elastic response of individual polyhedrons can be locally resolved, showing pronounced differences in stiffness between faces and vertexes of the structure, which makes these systems interesting as models for structurally similar colloidal scale objects such as viruses, where such effects are predicted but cannot be directly observed due to the smaller dimensions. Elastic constants of the wall material are estimated using shell and plate deformation models and are found to be a factor of 5 larger than those for neutral lipidic bilayers in the gel state. We discuss the molecular origins of this high stiffness.     

Regio- and stereoisomeric composition of the product mixture in the Diels–Alder reaction of dicyclopentadiene with bicyclononadiene: a NMR and DFT quantum chemical investigation

The regio- and stereoisomeric composition of the product mixture in the Diels–Alder reaction between dicyclopentadiene and bicyclononadiene is determined by 2D ¹H–¹³C HMQC and HMBC and ¹H 2D DQF COSY and NOESY NMR techniques. The absence in the experimental mixture of one of the expected products is explained by high-level quantum chemical density functional theory calculations.     

Liquid chromatographic/electrospray ionization mass spectrometric identification of the oxidation end‐products of trans‐resveratrol in aqueous solutions

trans‐Resveratrol (3,5,4′‐trihydroxystilbene) is a natural polyphenolic compound that exhibits antioxidant properties. Our study aimed at studying the HO ^. ‐induced oxidation of resveratrol (100 µmol.L^?1) in aerated aqueous solutions. Gamma radiolysis of water was used to generate HO ^. /O free radicals (I = 10 Gy.min^?1, dose = 400 Gy). Oxidation products were identified by direct infusion mass spectrometry and high‐performance liquid chromatography/mass spectrometry. For each product, structural elucidation was based on simple mass spectra, fragmentation spectra and deuterium/hydrogen exchange spectra; the comparison with mass spectra of synthetic products provided valuable information allowing the complete identification of the oxidation products. Four products resulting from the direct attack of HO ^. radicals towards resveratrol were identified respectively as piceatannol (trans‐3,5,3′,4′‐tetrahydroxystilbene), 3,5‐dihydroxybenzoic acid, 3,5‐dihydroxybenzaldehyde and 4‐hydroxybenzaldehyde. Copyright © 2010 John Wiley & Sons, Ltd.     

An Evaluation of High Performance Liquid Chromatography-UV for the Multi-Residue Analysis of Organophosphorous Pesticides in Environmental Water

Abstract

In this study isocratic high performance liquid chromatography in the reversed-phase mode (RP-8 or RP-18 column) with UV-detection (254 nm), was evaluated for the analysis, directly or after extraction, of organophosphorous pesticides in environmental water.

Fifteen pesticides were studied and good resolution was obtained for eight by direct analysis of a multi-residue water sample. Reproducibility in terms of retention times was found to be very good. Linear calibration curves were obtained down to 0.5ng/μL (3 ng injected) with a coefficient of variation near 10%. The limit of quantitation for direct analysis was found to be 0.5mg/L (3ng) in water. However, by extraction with methylene chloride or ethyl acetate and concentration of the extract some of the pesticides could be determined at 0.5 μg/L (ppb) in water.     

Development of a five-plex flow cytometric immunoassay for the simultaneous detection of six coccidiostats in feed and eggs

Coccidiostats are the only veterinary drugs still permitted to be used as feed additives to treat poultry for coccidiosis. To protect consumers, maximum levels for their presence in food and feed have been set by the European Union (EU). To monitor these coccidiostats, a rapid and inexpensive screening method would be a useful tool. The development of such a screening method, using a flow cytometry-based immunoassay, is described. The assay uses five sets of colour-coded paramagnetic microspheres for the detection of six selected priority coccidiostats. Different coccidiostats, with and without carrier proteins, were covalently coupled onto different bead sets and tested in combination with polyclonal antisera and with a fluorescent-labelled secondary antibody. The five optimal combinations were selected for this multiplex and a simple-to-use sample extraction method was applied for screening blank and spiked eggs and feed samples. A very good correlation (r ranging from 0.995 to 0.999) was obtained with the responses obtained in two different flow cytometers (Luminex 100 and FLEXMAP 3D). The sensitivities obtained were in accordance with the levels set by the EU as the measured limits of detection for narasin/salinomycin, lasalocid, diclazuril, nicarbazin (4,4'-dinitrocarbanilide) and monensin in eggs were 0.01, 0.1, 0.5, 53 and 0.1 μg/kg and in feed 0.1, 0.2, 0.3, 9 and 1.5 μg/kg, respectively.