Separation and characterisation of sphingoceramides by high-performance liquid chromatography-electrospray ionisation mass spectrometry

We developed a simple and reliable analytical method for the quantification and the characterization of ceramides extracted from biological samples by high-performance liquid chromatography (HPLC) coupled to electrospray ionisation tandem mass spectrometry (ESI/MS/MS). The chromatographic separation of analytes was carried out in a RP8 column, eluting with a methanol-water mixture in gradient elution mode. The separated lipids were detected by total ion monitoring and characterised by MS/MS spectra; quantitative analysis was performed by integrating the extracted ion peaks obtained in the negative ion mode. Good repeatability was obtained for retention time (0.3-2%), peak area ratio (A(S)/A(IS), 2-8%), as well as limit of detection (LOD, 5-26 pg) and quantification (LOQ, 13-53 pg). The method was validated for the analysis of N-palmitoyl-D-erythro-sphingosine (Cer16), N-stearoyl-D-erythro-sphingosine (Cer18), N-tetracosanoyl-D-erythro-sphingosine (N24:0, lignoceric ceramide, Cer24:0), and N-tetracos-15'-enoyl-D-erythro-sphingosine (N24:1, nervonic ceramide, Cer24:1), giving good results. Lipid mixtures, extracted from skin and epidermal cells, were analysed for their content of the studied ceramides.     

Selenium speciation by coupling vesicle mediated HPLC with off-line ETAAS and on-line focused microwave digestion HG-AAS detection

A novel High Performance Liquid Chromatography (HPLC) method for the separation of selenium species with specific detection by off-line Electrothermal Atomization Atomic Absorption Spectrometry (ETAAS) or on-line focused microwave digestion (MW) Hydride Generation Atomic Absorption Spectrometry (HG-AAS) is described. Vesicular mobile phases of the cationic surfactant didodecyldimethylammonium bromide (DDAB) have been evaluated for the liquid chromatographic separation of inorganic selenium (selenite and selenate) and different selenoaminoacids (selenocystine, selenomethionine and selenoethionine) on a C(18) reversed-phase column modified by DDAB molecules. The effects of different parameters (pH, buffer and vesicle concentrations) of the mobile phase on the retention times have been determined. The detection limit for selenium with the proposed off-line HPLC-ETAAS method has been found to be 5 microg/L of Se. The detection limit using HPLC-"on line" focused microwave digestion-HG-AAS has been found to be 1 microg/L of Se, with a precision (repeatability) better than +/- 5%. The latter proved to be an exceptional on-line real-time chromatographic detector for selenium speciation purposes.     

Surface plasmon resonance biosensor for genetically modified organisms detection

The development of a surface plasmon resonance (SPR) affinity biosensor based on DNA hybridisation is described. This biosensor has been applied to genetically modified organisms (GMOs) detection. Single stranded DNA (ssDNA) probes were immobilised on the sensor chip of an SPR device and the hybridisation between the immobilised probe and the complementary sequence (target) was monitored. The probe sequences were internal to the sequence of 35S promoter and NOS terminator which are inserted sequences in the genome of GMO regulating the transgene expression. The system has been optimised using synthetic oligonucleotides, then applied to real samples analysis. Samples, containing the transgenic target sequences, were amplified by polymerase chain reaction (PCR) and then detected with the SPR biosensor.     

Separation and analysis of glycyrrhizin, 18beta-glycyrrhetic acid and 18alpha-glycyrrhetic acid in liquorice roots by means of capillary zone electrophoresis

Glycyrrhizin is the main active compound of Glycyrrhiza glabra root extracts; according to recent studies, glycyrrhizin and its aglycon, glycyrrhetic acid, have interesting therapeutic properties. A new capillary electrophoretic method has been developed for the separation and quantification of glycyrrhizin, beta-glycyrrhetic acid and its isomer a-glycyrrhetic acid. Separation of the analytes was achieved in less than 3 min on a fused silica capillary, by injecting the samples at the short end of the capillary (effective length: 8.5 cm). The background electrolyte was composed of pH 10.0 carbonate buffer, methanol and ethylene glycol (80/10/10) and contained 0.4% beta-cyclodextrin; indomethacin was used as the internal standard. Diode array detection was used, with quantitative assays carried out at 254 nm. Linearity was found over the 5-200 and 2.5-100 microg mL(-1) concentration ranges for glycyrrhizin and glycyrrhetic acid, respectively. This method has been applied to the determination of the analytes in different matrices (liquorice roots and commercial confectionery products), and to the purity control of beta-glycyrrhetic acid obtained from the hydrolysis of glycyrrhizin. When analysing beta-glycyrrhetic acid and its epimer in roots, the samples were purified by means of a suitable solid-phase extraction (SPE) procedure with Oasis HLB cartridges, which granted good selectivity, eliminating matrix interference.     

A natural scalar field in the einstein gravitational theory

In a Riemannian space-time, the difference between the third-order tensor potentialH of the Riemann tensor (presented in a precedent paper) and the Lanczos generating function of the Weyl tensor is here shown to be characterized by a vectorV , obtained by contractionH . The significant role of such a vector, in the context of general relativity, is then discussed. Particular attention is paid to the scalar potential which characterizes the irrotational part ofV : such a scalar field satisfies a space-time wave equation of the Poisson type. Weak fields are also considered: in the particular case of a static metric, the scalar is found to be proportional to the classic Newtonian potential.This work was done in the sphere of activity of the C.N.R. Groups for mathematical research.     

Zur Ermittlung der Äthylen-Sequenzlängenverteilung und der chemischen Uneinheitlichkeit in Copolymeren mit Propylen durch Pyrolyse-Gas-Chromatographie

Pyrolysis coupled with gas chromatography has been applied to investigate the sequence distribution in copolymers of ethylene and propylene. The copolymers analyzed have been prepared with soluble Ziegler-Natta-catalysts and show different degrees of crystallinity. In one series of samples the methyl group of the propylene units has been labeled with tritium. It was then possible to detect carbons by flame ionization detector and simultaneously the methyl groups by the radio-detector in parallel. Within the errors of these methods the results from both detectors are consistent with the calculations from kinetics. The accuracy of the data is best in copolymers with long ethylene sequences. By GPC fractionation a heterogeneity in the chemical composition can be observed. One component of the copolymers is of high molecular weight, while the other has a molecular weight of less than 1000 and is very rich in ethylene. The influence of chemical heterogeneity on sequence distribution derived from kinetics and pyrolysis has been discussed. A mechanism in which chains with different chemical composition are growing from different species of the catalyst has been proposed. The experimental results show that the heterogeneity is not critical for the sequence analysis by kinetics and pyrolysis.     

Hydrophobic Molecular Similarity from MST Fractional Contributions to the Octanol/water Partition Coefficient

Summary The use of a recently proposed hydrophobic similarity index for the alignment of molecules and the prediction of their differences in biological activity is described. The hydrophobic similarity index exploits atomic contributions to the octanol/water transfer free energy, which are evaluated by means of the fractional partitioning scheme developed within the framework of the Miertus-Scrocco-Tomasi continuum model. Those contributions are used to define global and local measures of hydrophobic similarity. The suitability of this computational strategy is examined for two series of compounds (ACAT inhibitors and 5-HT₃ receptor agonists), which are aligned to maximize the global hydrophobic similarity using a Monte Carlo-simulated protocol. Indeed, the concept of local hydrophobic similarity is used to explore structure–activity relationships in a series of COX-2 inhibitors. Inspection of the 3D distribution of hydrophobic/hydrophilic contributions in the aligned molecules is valuable to identify regions of very similar hydrophobicity, which can define pharmacophoric recognition patterns. Moreover, low similar regions permit to identify structural elements that modulate the differences in activity between molecules. Finally, the quantitative relationships found between the pharmacological activity and the hydrophobic similarity index points out that not only the global hydrophobicity, but its 3D distribution, is important to gain insight into the activity of molecules. J.M.M. and S.P. have contributed equally to this study.     

CsOH-promoted epoxide ring-opening with phosphines: mild and efficient synthesis of monohydroxyphosphines

A mild and convenient synthesis of monohydroxyphosphines has been achieved by epoxide ring-opening using primary or secondary phosphines in the presence of cesium hydroxide, 4 Å molecular sieves and DMF at room temperature. These reaction conditions were found to be highly regio- and stereoselective producing various monohydroxyphosphines exclusively in moderate to high yields.     

CCR5 antagonists: 3-(pyrrolidin-1-yl)propionic acid analogues with potent anti-HIV activity

[reaction: see text] A novel approach to alpha,alpha-disubstituted-beta-amino acids (beta(2,2)-amino acids) was employed in the synthesis of a series of 3-(pyrrolidin-1-yl)propionic acids possessing high affinity for the CCR5 receptor and potent anti-HIV activity. The rat pharmacokinetics for these new analogues featured higher bioavailabilities and lower rates of clearance as compared to cyclopentane 1.