Liquid chromatographic method for nicarbazin in broiler feeds and premixtures: development, validation, and interlaboratory study

A reversed-phase liquid chromatography method for nicarbazin in broiler feeds and premixtures was developed, validated, and interlaboratory studied. The extraction solvent was an acetonitrile-methanol (1 + 1) mixture. For feedingstuffs, water was also added. The 4,4'-dinitrocarbanilide moiety of nicarbazin was detected at a wavelength of 350 nm. Recovery was > or =87%. At 20 mg/kg, the repeatability was 0.7% and the within-laboratory reproducibility was 2.7%. The limit of determination was <20 mg/kg. Other feed additives did not interfere in the assay that proved to be applicable to broiler feeds from different European Union countries. In an interlaboratory study, 4 positive broiler feeds, 1 blank pig feed, and 1 broiler premixture were analyzed by 19 laboratories using the method developed in this study. The relative standard deviation for repeatability (RSDr) of the feedingstuffs (20-240 mg/kg) varied between 2.6 and 10.2%. The HORRAT ranged between 0.70 and 1.22. Recoveries were 91-108%. Three laboratories detected small signals in the blind blank samples, ranging from 0.4 to 2 mg/kg. For the premixture, acceptable results for reproducibility could only be obtained after the sample weight and volume of extraction had been doubled. To avoid excessive dilution of the extracts, the range of the calibration curve had also been doubled. With this modified method, the RSDr was 5.7% and the HORRAT was 1.95 (10 laboratories).     

The Kemp elimination in membrane mimetic reaction media. Probing catalytic properties of cationic vesicles formed from a double-tailed amphiphile and linear long-tailed alcohols or alkyl pyranosides

Vesicles formed from synthetic, double-tailed amphiphiles are often used as mimics for biological membranes. However, biological membranes are a complex mixture of various compounds. In the present paper we describe a first attempt to study the importance of additives on vesicular catalysis. The rate-determining deprotonation of 5-nitrobenzisoxazole (Kemp elimination) by hydroxide ion is efficiently catalysed by vesicles formed from dimethyldi-n-octadecylammonium chloride (C(18)C(18)(+)) as a result of (partial) dehydration of the reactants (especially the hydroxide ion) at the vesicular binding sites. Gradual addition of linear alcohols, such as n-decanol (C(10)OH), n-octadecanol (C(18)OH) and batyl alcohol (C(18)GlyOH) leads to a decrease in the observed catalysis. By contrast, gradual addition of oleyl alcohol, n-dodecyl-beta-glucoside (C(12)Glu) and n-dodecyl-beta-maltoside (C(12)Mal) leads to an increase in the observed catalysis. A detailed kinetic analysis, taking into account substrate binding site polarities, counterion binding percentages and binding affinity of the kinetic probe, suggests that the catalytic changes depend strongly on subtle changes in the structure of the additive. Whereas the C(12)Glu-induced effect can be explained by an increase in the vesicular rate constant, the effect of C(12)Mal can only be explained by an increase in the binding constant of the kinetic probe. However, for these pyranoside-containing vesicles others factors, such as a more extensive dehydration of the hydroxide ion, and micelle formation have to be considered. For the linear alcohols, besides a decrease in the counterion binding, changes in the vesicular rate constant and the binding constant should be taken into account. These two parameters change to a different extent for the different alcohols. The kinetic analysis is supported by differential scanning calorimetry (DSC), E(T)(30) absorbance data and Nile Red, Laurdan, ANS and pyrene fluorescence measurements.The overall kinetic results are illustrative for the highly complex mix of factors which determines catalytic effects on reactions occurring in biological cell membranes.     

List of participants

List of participants     

Fluorescence biosensing micropatterned surfaces based on immobilized human acetylcholinesterase

Human acetylcholinesterase (AChE) is a widely studied target enzyme in drug discovery for Alzheimer’s disease (AD). In this paper we report evaluation of the optimum structure and chemistry of the supporting material for a new AChE-based fluorescence sensing surface. To achieve this objective, multilayered silicon wafers with spatially controlled geometry and chemical diversity were fabricated. Specifically, silicon wafers with silicon oxide patterns (SiO₂/Si wafers), platinum-coated silicon wafers with SiO₂ patterns (SiO₂/Pt/Ti/Si wafers), and Pt-coated wafers coated with different thicknesses of TiO₂ and SiO₂ (SiO₂/TiO₂/Pt/Ti/Si wafers) were labelled with the fluorescent conjugation agent HiLyte Fluor 555. Selection of a suitable material and the optimum pattern thickness required to maximize the fluorescence signal and maintain chemical stability was performed by confocal laser-scanning microscopy (CLSM). Results showed that the highest signal-to-background ratio was always obtained on wafers with 100 nm thick SiO₂ features. Hence, these wafers were selected for covalent binding of human AChE. Batch-wise kinetic studies revealed that enzyme activity was retained after immobilization. Combined use of atomic-force microscopy and CLSM revealed that AChE was homogeneously and selectively distributed on the SiO₂ microstructures at a suitable distance from the reflective surface. In the optimum design, efficient fluorescence emission was obtained from the AChE-based biosensing surface after labelling with propidium, a selective fluorescent probe of the peripheral binding site of AChE.

A simple and universal method for the separation and identification of phospholipid molecular species

One of the major challenges in lipidomics is to obtain as much information about the lipidome as possible. Here, we present a simple yet universal high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS) method to separate molecular species of all phospholipid classes in one single run. The method is sensitive, robust and allows lipid profiling using full scan mass spectrometry, as well as lipid class specific scanning in positive and negative ionisation mode. This allows high-throughput processing of samples for lipidomics, even if different types of MS analysis are required. Excellent separation of isobaric and even isomeric species is achieved, and original levels of lyso-lipids can be determined without interference from lyso-lipids formed from diacyl species by source fragmentation. As examples of application of this method, more than 400 phospholipid species were identified and quantified in crude phospholipid extracts from rat liver and the parasitic helminth Schistosoma mansoni.     

Dynamic chirality, chirality transfer and aggregation behaviour of dithienylethene switches

The synthesis and characterisation of a series of chiral and achiral low molecular weight organogelators (LMWGs) based on bis-amide substituted dithienylethene photochromic switches is reported. The LMWGs gelate a range of solvents depending on the specific functionalisation of the hydrogen bonding amide groups. In mixtures of chiral and achiral LMWGs the stereochemical outcome of the chiral aggregation is determined by the chiral LMWG molecules in most cases. However, for the first time we demonstrate that the stereochemical outcome of the aggregation can be influenced by the achiral LWMG molecules in some cases. Furthermore specific π-π (and/or van der Waals) interactions of chiral LMWGs 1-3o with the solvent allow the solvent to influence the control of chirality of aggregation. This influence of the solvent has a dramatic effect on whether four- or two-gel states are available.     

Molecular characterization of organically-bound sulfur in crude oils. A feasibility study for the application of Raney Ni desulfurization as a new method to characterize crude oils

Five crude oils with varying sulfur contents (0.1 – 4.7%) were characterized on a molecular level for organically-bound sulfur. Aromatic fractions were analyzed by GC-(MS) and asphaltene and polar fractions were analyzed by flash pyrolysis-GC-(MS). The polar fractions were also desulfurized with Raney Ni and the hydrocarbons formed were analyzed by GC-MS. Major sulfur compounds in the aromatic fractions were identified as alkylbenzo- and alkyldiben-zothiophenes. The flash pyrolyzates of the asphaltene contained alkylthiophenes and alkylbenzothiophenes as major compounds, depending on the thermal maturity of the oil. Generally, the sulfur-rich crude oils contained relatively more sulfur compounds. The flash pyrolyzates of polar fractions contained a variety of sulfur compounds (alkylthiolanes, alkylthianes, terpenoid sulfides, alkylbenzothiophenes) with substantial differences between different crude oils. Raney Ni desulfurization of the polar fraction yielded hydrocarbons dominated by n-alkanes, but isoprenoid alkanes, n-alkylcyclohexanes, mid-chain methylalkanes, tricyclic terpanes, hopanes and steranes were also present. These hydrocarbons show a potential to fingerprint crude oils since their distribution patterns are more characteristic than those of the hydrocarbons present in the saturated hydrocarbon fraction.     

Copper(0) in the Ullmann heterocycle-aryl ether synthesis of 4-phenoxypyridine using multimode microwave heating

The action of nanoparticulate copper catalysts with a mean particle size of 10 nm in the Ullmann ether synthesis is reported using multimode microwave heating and employing stable chloropyridine salts and unactivated phenol, with stabilized copper nanoparticles outperforming other copper catalysts in terms of stability and reusability.