Isolation from pig liver microsomes, identification by electrospray tandem mass spectrometry and in vitro immunosuppressive activity of a rapamycin tris-epoxide metabolite

It was demonstrated that rapamycin is metabolized in vitro by pig liver microsomes under the influence of the cytochrome P450-dependent mixed function oxygenase system to a rapamycin tris-epoxide metabolite, as demonstrated by electrospray tandem mass spectrometry. The in vitro immunosuppressive activity of this metabolite was found to be lower than that of rapamycin, probably because the rapamycin effector sector was structurally modified. The effector region of rapamycin was recognized to include the conjugated double bonds of this compound and metabolic reactions affecting this region may change the binding affinity of the rapamycin-FKBP binary complex towards another pharmacological receptor bound to the binary complex. Moreover, metabolic modifications in the effector region are probably able to induce a change in the binding affinities of the rapamycin-FKBP binary complex, including the pipecolic acid moiety and the lactone function of the parent drug.     

In vitro immunosuppressive activity of tacrolimus dihydrodiol precursors obtained by chemical oxidation and identification of a new metabolite of SDZ-RAD by electrospray and electrospray-linked scan mass spectrometry

Different tacrolimus epoxides and dihydrodiol epoxides arising from the chemical oxidation of the parent drug are described. Open-chain tautomeric forms involving the lactone function were identified for the tacrolimus epoxides. Moreover, the identification by electrospray and electrospray linked scan mass spectrometry of an SDZ-RAD C16-C27 O-demethyl 17, 18-19, 20-21, 22 tris-epoxide new metabolite isolated from pig liver microsomes is reported. The in vitro immunosuppressive activity, using mixed lymphocyte reactions of the two macrolide reported oxidation compounds are discussed.     

Determination of carphedon in human urine by solid-phase microextraction using capillary gas chromatography with nitrogen-phosphorus detection

Carphedon is a phenyl derivative of nootropil and is effective in increasing physical endurance and cold resistance, and is used for amnesia treatment. Carphedon was extracted from human urine samples by solid-phase microextraction with a 65 microns carbowax-divinylbenzene-coated fiber. This analysis was performed by using capillary gas chromatography with nitrogen-phosphorus detection and optimized at pH 9.6, 30% NaCl, immersion time 10 min and desorption in the GC injector at 250 degrees C for 3 min. The regression equation for carphedon showed good linearity in the range from 0.1 to 10 micrograms ml-1 for human urine samples. The limit of detection was 0.01 microgram ml-1. The developed method is more sensitive and simpler in sample preparation than liquid-liquid extraction and can be applied to doping analysis for stimulants.     

Isolation from rat urine and human liver microsomes and identification by electrospray and nanospray tandem mass spectrometry of new malagashanine metabolites

Malagashanine has been isolated from indigenous madagascan Strychnos myrtoides alkaloids used traditionally to treat malaria. This alkaloid was found to enhance the action of chloroquine against chloroquine-resistant strains of Plasmodium falciparum when combined with classical antimalarial drugs (chloroquine, quinine). The present study was carried out in order to investigate by electrospray mass and tandem mass spectrometry and NMR spectroscopy the structure of two new metabolites isolated from rat urine and human liver microsomes. We were able to demonstrate the presence of two new metabolites of malagashanine corresponding to a malagashanine N-demethylated metabolite and to the oxidation of malagashanine in the alpha-position of the N-methyl group to produce a carbinolamine function. The latter metabolite may be subject to ring and open-chain tautomerism effects and dimeric species were detected in the electrospray mass spectrum.     

Isolation from pig liver microsomes, identification by tandem mass spectrometry and in vitro immunosuppressive activity of an SDZ-RAD 17,18,19,20,21,22-tris-epoxide

Macrolide immunosuppressive drugs such as tacrolimus (FK506) and sirolimus (rapamycin) are compounds largely used in modern immunosuppressive therapy and considered as powerful immunosuppressive agents. Some of these molecules are still under clinical development as, for example, SDZ-RAD (40-O-(2-hydroxyethyl)rapamycin), an immunosuppressive drug closely related to rapamycin. SDZ-RAD has a molecular mass of 957.57 Da (C53H83NO14) and shares the same common intracellular receptor as tacrolimus, the FK-506 binding protein (FKBP-12). SDZ-RAD exerts its pharmacological effect by binding to a different effector protein, inhibits the S6p 70-kinase and interrupts a different signal transduction pathway than tacrolimus. Both SDZ-RAD and rapamycin are metabolized mainly by the cytochrome P-450 3A4-dependent mixed function oxygenase enzyme system to hydroxylated and demethylated metabolites. We describe here the isolation from pig liver microsomes of a novel SDZ-RAD metabolite identified by electrospray tandam mass spectrometry as a new SDZ-RAD 17,18,19,20,21,22-tris-epoxide metabolite. The in vitro immunosuppressive activity as measured by the mixed lymphocyte reaction is more or less comparable to that of SDZ-RAD, although its pharmacological mode of action may be different from that classically described for rapamycin.     

Capillary electrophoretic competitive immunoassay with laser-induced fluorescence detection for methionine-enkephalin

Immunoassays are commonly used in bioresearch for the detection and quantification of small proteins and macromolecules in biological fluids and other complex matrices. In this report, a competitive immunoassay using capillary electrophoresis (CE) with laser-induced fluorescence was developed for methionine-enkephalin (ME). The method is based on the competitive reaction between the ME and fluorescein conjugated ME (ME-F) with anti-ME antibody, capillary electrophoresis separation of the ME-antibody bound and free ME-F, followed by the laser-induced fluorescence detection of the fluorescent species. With the optimized separation conditions, it was possible to separate the antibody bound and free fluorescien conjugated ME by a capillary electrophoresis-laser-induced fluorescence (CE-LIF) analysis using an uncoated fused-silica capillaries. The results concluded that the assay specificity, selectivity and accuracy were excellent.     

Synthesis of titanium dioxide in O2/Ar/SO2/TiCl4 microwave torch plasma and its band gap narrowing

Titanium dioxide (TiO₂) nanoparticles were synthesized directly by injecting gas-phase titanium tetrachloride (TiCl₄) bubbled with a mixture of Ar and SO₂ into a 2.45 GHz microwave torch plasma operated in O₂ gas at atmospheric pressure. The absorption edge of the as-produced TiO₂ powders in the UV–Visible spectrum shifted from 457 nm to 483 nm as the SO₂ gas content in the mixture gas carrying the TiCl₄ was increased. According to X-ray photoelectron spectroscopy (XPS) and UV–Visible spectroscopy, the band gap narrowing was due to the incorporation of sulphur (S) species from the Ar and SO₂ mixture in the oxygen microwave torch plasma into the O site of TiO₂.     

Ballast Water Treatment Test at Pilot-Scale Using an Underwater Capillary Discharge Device

We demonstrated a pilot-scaled underwater plasma discharge device for the treatment of ballast water (5 ton/h). Phytoplankton and microorganisms in the ballast water were successfully removed using underwater plasma with capillary electrodes. Microorganisms between 10 and 50 μm in the ballast water were eliminated with an efficiency of 99.9% as well as heterotrophic bacteria with an efficiency of 92.7%.