Application of micro-thin-layer chromatography as a simple fractionation tool for fast screening of raw extracts derived from complex biological, pharmaceutical and environmental samples

The main goal of present paper is to demonstrate the separation and detection capability of micro-TLC technique involving simple one step liquid extraction protocols of complex materials without multi-steps sample pre-purification. In the present studies target components (cyanobacteria pigments, lipids and fullerenes) were isolated from heavy loading complex matrices including spirulina dried cells, birds’ feathers and fatty oils as well as soot samples derived from biomass fuel and fossils-fired home heating systems. In each case isocratic separation protocol involving less that 1 mL of one component or binary mixture mobile phases can be completed within time of 5–8 min. Sensitive detection of components of interest was performed via fluorescence or staining techniques using iodine or phosphomolybdic acid. Described methodology can be applied for fast fractionation or screening of whole range of target substances as well as chemo-taxonomic studies and fingerprinting of complex mixtures, which are present in raw biological or environmental samples.     

Development of an efficient protein phosphatase-based colorimetric test for okadaic acid detection

Okadaic acid (OA), responsible for gastrointestinal problems, inhibits protein phosphatase 2A (PP2A). Therefore, the inhibition exerted by the toxin on PP2A could be used to detect the presence of OA in aqueous solution and in shellfish sample.In this work, two commercial PP2As (from ZEU Immunotec and Millipore) and one produced by molecular engineering (from GTP Technology) were tested. Enzymes were used in solution and also immobilized within a polymeric gel. In solution, best performances were obtained using PP2A purchased from ZEU Immunotec (Spain). OA was detected in aqueous solution in concentration as low as 0.0124 μg L⁻¹ using the enzyme from ZEU Immunotec whereas the detection limits were 0.47 μg L⁻¹ and 0.123 μg L⁻¹ with PP2As from Millipore and GTP Technology, respectively. Considering that the immobilization step contributes to stabilize the PP2A activity, enzymes were entrapped within a photopolymer and an agarose gel. These different polymeric matrices were optimized, tested and compared. Agarose gel seems to be a good alternative to the photopolymer largely used in our group. For instance, the IC₅₀ value obtained with the test based on PP2A from ZEU Immunotec immobilized within an agarose gel was 1.98 μg L⁻¹. This value was 1.8-fold lower than those obtained with the photopolymer test using the same enzyme. The proposed test is sensitive, fast and does not require expensive equipment. To evaluate the efficiency of the assay, PP inhibition tests based on PP2A from ZEU Immunotec in solution or immobilized within a gel were used for OA detection in contaminated shellfish.     

Are antibiotic screening approaches sufficiently adequate? A proficiency test

A proficiency test including the screening analysis of antibiotics in beef using cryogenicly minced materials was organized by RIKILT in 2009. The test included blank beef samples and beef samples spiked with either flumequine or a combination of lincomycin and spectinomycin around the maximum residue limits [1]. The suitability of the materials was demonstrated with a homogeneity and a stability study. This study showed that cryogenically minced spiked muscle material is suited for proficiency tests aiming at the screening and the confirmatory analysis.Of the 26 participants, 23 carried out their in-house screening approach involving microbial, biochemical or instrumental methods, or a combination of these to cover the broad range of antibiotic groups. The false negative rate was 73% for microbial methods, 50% for biochemical and 22% for instrumental methods. These results indicate that substantial effort is needed to improve screening approaches and that more regular proficiency tests are needed to reveal the shortcomings in the currently applied screening methods.     

SANS and DSC study of water distribution in epoxy-based hydrogels

Distribution of water in stoichiometric hydrophilic epoxy network swollen in heavy water to different degrees (epoxy-based hydrogels) at 25 °C has been investigated by small-angle neutron scattering (SANS) and differential scanning calorimetry (DSC). Nanophase separated structure of the hydrogels consisting of water-rich and water-poor domains was revealed by SANS. Two regimes for hydrogel structure were found: (a) at low water content hydrogel consists of isolated water-rich domains dispersed in continuous water-poor phase and (b) at high water content the water-rich domains form another continuous phase. Isosbestic point of scattering curves was found by SANS in the latter region and attributed to conservation of Porod’s length of the nanophase separated structure. Thermal properties of the system are qualitatively different in the two regions: in the former one the glass transition temperature decreases with growing water content while in the latter one it remains constant. Percolation threshold separating both regimes is reflected in a jump of glass transition temperature and inversion of the dependence of the specific heat difference at glass transition.     

First identification of dimethoxycinnamic acids in human plasma after coffee intake by liquid chromatography-mass spectrometry

There is a substantial amount of published literature on the bioavailability of various coffee components including the most abundant metabolites, caffeic and ferulic acids. Surprisingly, to date, the appearance of dimethoxycinnamic acid derivatives in humans has not been reported despite the fact that methylated form of catechol-type polyphenols could help maintain, modify or even improve their biological activities. This study reports an LC-MS method for the detection of dimethoxycinnamic acid in human plasma after treatment with an esterase. Liquid chromatography, including the combination of methanol and acetonitrile as organic eluent, was optimized to resolve all interferences and enable reliable detection and identification of 3,4-dimethoxycinnamic and 3,4-dimethoxy-dihydrocinnamic acids. In addition to the good mass accuracy achieved (better than 5 ppm), tandem mass spectrometric and co-chromatography experiments further confirmed the identity of the compounds. The optimized method was applied to analyze samples obtained immediately, 1 and 10 h after coffee ingestion. The results show that in particular 3,4-dimethoxycinnamic acid appears in high abundance (～380 nM at 60 min) in plasma upon coffee intake, indicating that it is important to consider these derivatives in future bioavailability and bioefficacy studies.     

Hydrophilic interaction liquid chromatography in food analysis

The use of hydrophilic interaction liquid chromatography (HILIC) in food analysis in the last decade is reviewed. The HILIC mechanism is briefly discussed, but main emphasis is put on the use of HILIC for separation of different food matrices. The food matrices are divided into food of animal origin and related products, vegetables, fruits and related compounds, and other food-related matrices. A list on important applications is provided for each category including experimental conditions and a brief summary of the results. The 100 references included will provide the reader a comprehensive overview and insight into HILIC applications to food analysis.     

Comparison of epithelial and fibroblastic cell behavior on nano/micro-topographic PCL membranes produced by crystallinity control

In this study, the relationship between the cellular morphology and the material surface topography was investigated. Poly(ε-caprolactone) (PCL) membranes were prepared in a wide range of surface wettabilities by means of crystallinity-controlled solvent casting process. Membrane surfaces were characterized by atomic force microscope (AFM), scanning electron microscope (SEM), and static/dynamic water contact angle measurements. It was found that solvent evaporation and non-solvent (methanol) addition to the solvent (THF) are the most decisive parameters to change the surface topography. The non-solvent addition and the decrease in solvent evaporation temperature from room temperature to -20 °C caused increased polymeric chain mobility and crystallization time. Such changes in crystallization parameters led to the formation of micro/nano-sized features on the membrane. Cell culture studies indicated that in contrast to Madin Darby kidney (MDBK) epithelial cells, L929 mouse fibroblast preferred rough and porous surfaces.     

One‐step ring‐closure procedure for 4,5‐dihydro‐1,3‐thiazino[5,4‐b]indole derivatives with Lawesson's reagent. The fifth dihydro‐1,3‐thiazino[b]indole isomer

We report a convenient approach for the synthesis of a new ring system: 4,5‐dihydro‐1,3‐thiazino[5,4‐b]indoles. The procedure involves the use of Lawesson's reagent in the presence of silica to achieve the one‐step ring‐closure reactions of 2‐benzoylamino‐3‐hydroxymethylindole intermediates to furnish 4,5‐dihydro‐2‐aryl‐1,3‐thiazino[5,4‐b]indoles. 2‐Phenylimino‐1,3‐thiazino[5,4‐b]indoles were obtained via the corresponding 3‐phenylthiourea‐2‐carboxylic acid ester derivatives by chemoselective reduction of the ester group, followed by ring closure under acidic conditions. The structures of the novel products were elucidated by IR, ¹H‐NMR, and ¹³C‐NMR spectroscopy, including 2D‐HMQC, 2D‐HMBC, and DEPT measurements. J. Heterocyclic Chem., (2011).     

Influence of mixed thiolate/thioether versus dithiolate coordination on the accessibility of the uncommon +I and +III oxidation states for the nickel ion: an experimental and computational study

Sulfur-rich nickel metalloenzymes are capable of stabilizing Ni(I) and Ni(III) oxidation states in catalytically relevant species. In an effort to better understand the structural and electronic features that allow the stabilization of such species, we have investigated the electrochemical properties of two mononuclear N(2)S(2) Ni(II) complexes that differ in their sulfur environment. Complex 1 features aliphatic dithiolate coordination ([NiL], 1), and complex 2I is characterized by mixed thiolate/thioether coordination ([NiL(Me)]I, 2I). The latter results from the methylation of a single sulfur of 1. The X-ray structure of 2I reveals a distorted square planar geometry around the Ni(II) ion, similar to what was previously reported by us for 1. The electrochemical investigation of 1 and 2(+) shows that the addition of a methyl group shifts the potentials of both redox Ni(II)/Ni(I) and Ni(III)/Ni(II) redox couples by about 0.7 and 0.6 V to more positive values. Through bulk electrolyses, only the mononuclear dithiolate [Ni(I)L](-) (1(-)) and the mixed thiolate/thioether [Ni(III)L(Me)](2+) (2(2+)) complexes were generated, and their electronic properties were investigated by UV-vis and EPR spectroscopy. For 1(-) (Ni(I), d(9) configuration) the EPR data are consistent with a d(x(2))(-)(y(2)) based singly occupied molecular orbitals (SOMOs). However, DFT calculations suggest that there is also pronounced radical character. This is consistent with the small g-anisotropy observed in the EPR experiments. The spin population (Mulliken analysis) analysis of 1(-) reveals that the main contribution to the SOMO (64%) is due to the bipyridine unit. Time dependent density functional theory (TD-DFT) calculations attribute the most prominent features observed in the electronic absorption spectrum of 1(-) to metal to ligand charge transfer (MLCT) transitions. Concerning 2(2+), the EPR spectrum displays a rhombic signal with g(x) = 2.236, g(y) = 2.180, and g(z) = 2.039 in CH(3)CN. The g(iso) value is larger than 2.0, which is consistent with metal based oxidation. The unpaired electron (Ni(III), d(7) configuration) occupies a Ni-d(z(2)) based molecular orbital, consistent with DFT calculations. Nitrogen hyperfine structure is observed as a triplet in the g(z) component of the EPR spectrum with A(N) = 51 MHz. This result indicates the coordination of a CH(3)CN molecule in the axial position. DFT calculations confirm that the presence of a fifth ligand in the coordination sphere of the Ni ion is required for the metal-based oxidation process. Finally, we have shown that 1 exhibits catalytic reductive dehalogenation activity below potentials of -2.00 V versus Fc/Fc(+) in CH(2)Cl(2).     

Ultrafast dynamics of aniline in the 294-234 nm excitation range: the role of the πσ∗ state

The ultrafast relaxation of jet-cooled aniline was followed by time-resolved ionization, after excitation in the 294-234 interval. The studied range of energy covers the absorption of the two bright ππ? excitations, S(1) and S(3), and the almost dark S(2) (πσ?) state. The employed probe wavelengths permit to identify different ultrafast time constants related with the coupling of the involved electronic surfaces. A τ(1) = 165 ± 30 fs lifetime is attributed to dynamics along the S(2) (πσ?) repulsive surface. Other relaxation channels as the S(1)→S(0) and S(3)→S(1) internal conversion are also identified and characterized. The work provides a general view of the photophysics of aniline, particularly regarding the role of the πσ? state. This state appears as minor dissipation process due to the ineffective coupling with the bright S(1) and S(3) states, being the S(1)→S(0) internal conversion the main non-radiative process in the full studied energy range. Additionally, the influence of the off-resonance adiabatic excitation of higher energy electronic states, particularly S(3), is also observed and discussed.