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At least 343 records · Page 19Linked to original sources
PubMed1995

Transbilayer distribution of phosphatidylcholine and phosphatidylethanolamine in the vacuolar membrane of Acer pseudoplatanus cells.

The distribution of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) among the outer and inner monolayers of the vacuolar membrane of Acer pseudoplatanus was investigated using isolated vacuoles, chemical labelling agents (trinitrobenzene-sulfonate and fluorescamine), phospholipase A2 from bee venom, phospholipase C and phospholipase D. Treatments were performed with intact or sonicated vacuoles. Analysis of the transbilayer distribution of PC and PE in the vacuolar membrane of Acer was limited by phospholipid fractions which were inaccessible to the probes. Lipid-protein interactions and modification of the surface charge and surface pressure in the membrane layers during treatments may obviously exert a strong influence on labelling or hydrolysis of membrane phospholipids. However, simultaneous treatments carried out with phospholipase A2 and phospholipase C show that PE is approximately 20% more abundant in the outer monolayer than in the inner monolayer and PC is equally distributed between both leaflets of tonoplast. Compared to the phospholipids asymmetrical distribution observed in plasma membrane of erythrocyte, the vacuolar membrane of Acer is not characterized by a marked asymmetrical distribution of its major phospholipids.

Cells, Cultured↗
PubMed1994

Quantitative determination of CGP 53,437, a new HIV protease inhibitor, in plasma by high-performance liquid chromatography and fluorescence detection.

A specific and sensitive liquid chromatographic assay for CGP 53,437 (I), a potent HIV protease inhibitor, is described. The method is based on a deproteinization step, followed by a liquid-liquid extraction with diisopropyl ether. Then a deprotection step of the primary amine and derivatization using fluorescamine is performed. Chromatography is achieved by isocratic elution with a mobile phase of 63 mM borax buffer (pH 9)-acetonitrile (58:42, v/v). The flow-rate of the mobile phase is 1 ml/min. The derivatives of compound I and its internal standard CGP 54,451, II, fluoresce at 480 nm on excitation at 395 nm. The limit of quantitation which is the lowest concentration of the analyte that can be measured with a coefficient of variation and a deviation from theory of less than 20%, was 5 nmol/l plasma. The analyte is stable for at least seven months in spiked human plasma samples. It is also stable after freezing and thawing cycles. Different human plasma sources and plasma samples from three different species (dog, marmoset, and rat) were tested and no interferences from plasma constituents was observed.

Animals↗
PubMed1992

LC assay for salmon calcitonin in aerosol formulations using fluorescence derivatization and size exclusion chromatography.

A highly sensitive LC procedure was developed that utilizes fluorescence derivatization and detection coupled with size exclusion chromatography for the analysis of salmon calcitonin in salmon calcitonin aerosols. The LC procedure uses fluorescamine derivatization to label the primary amino groups of the peptide. The derivatization procedure is completely automated by an autosampler capable of pre-column mixing. Size exclusion chromatography is performed using a Supelco G2000 SWXL column. The method can be used to assay the amount of salmon calcitonin delivered per actuation of an aerosol unit. The procedure is simple, accurate, and precise and can detect as little as 2 ng ml-1 concentrations of salmon calcitonin.

Aerosols↗
PubMed1995

Micellar-enhanced synchronous-derivative fluorescence determination of derivatized folic acid in pharmaceutical preparations.

A sensitive and inexpensive micellar-enhanced synchronous-derivative spectrofluorimetric method is described for the determination of folic acid in pharmaceutical preparations. The method is based upon derivatization of the vitamin with fluorescamine in acid solution and enhancement of the fluorescence with a surfactant. Linear fluorimetric analytical curves were obtained for folic acid concentrations of 0.05-6 micrograms ml-1 (detection limit 0.012 micrograms ml-1); the RSD for 1 micrograms ml-1 of folic acid was 2.82%. The method has been applied to the determination of folic acid synthetic mixtures and pharmaceutical preparations.

Detergents↗
PubMedDate not supplied

Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme).

The extracellular initiation protein (IP; spore germination enzyme) produced by Clostridium perfringens was further purified and characterized. IP hydrolysed spore cortical fragments with the release of free amino groups. End group analysis of hydrolysed fragments indicated the presence of N-terminal alanine but no reducing sugars. Molecular weight analysis of IP- and lysozyme-treated fluorescamine-labelled cortical fragments indicated that IP acts only on peptidoglycan chains containing cross-linked peptide subunits. IP failed to hydrolyse a number of nitrophenyl-conjugated glucopyranosides and galactopyranosides. The results indicate that IP is an N-acetylmuramyl-L-alanine amidase.

Amidohydrolases↗
PubMed2006

Metal contaminants promote degradation of lipid/DNA complexes during lyophilization.

Oxidation reactions represent an important degradation pathway of nucleic acid-based pharmaceuticals. To evaluate the role of metal contamination and chelating agents in the formation of reactive oxygen species (ROS) during lyophilization, ROS generation and the stability of lipid/DNA complexes were investigated. Trehalose-containing formulations were lyophilized with different levels of transition metals. ROS generation was examined by adding proxyl fluorescamine to the formulations prior to freeze-drying. Results show that ROS were generated during lyophilization, and both supercoil content and transfection rates decreased as the levels of metal-induced ROS increased. The experiments incorporating chelators demonstrated that some of these agents (e.g., DTPA, desferal) clearly suppress ROS generation, while others (e.g., EDTA) enhance ROS. Surprisingly, there was not a strong correlation of ROS generated in the presence of chelators with the maintenance of supercoil content. In this study, we demonstrated the adverse effects of the presence of metals (especially Fe(2+)) in nonviral vector formulations. While some chelators attenuate ROS generation and preserve DNA integrity, the effects of these additives on vector stability during lyophilization are difficult to predict. Further study is needed to develop potent formulation strategies that inhibit ROS generation and DNA degradation during lyophilization and storage.

Animals↗
PubMed2005

High performance liquid chromatography post-column chemiluminescence determination of sulfonamide residues in milk at low concentration levels using bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate as chemiluminescent reagent.

The determination of seven sulfonamides by means of HPLC with chemiluminescence detection is proposed for the first time. The analytes are derivatized with fluorescamine, separated and subsequently they participate in the post-column chemiluminescence (CL) peroxyoxalate system using imidazole as a catalyst. Among the different peroxyoxalates tested, bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl] oxalate provides higher sensitivities and stabilities, avoiding precipitation problems. A rigorous optimization of the significant variables by means of experimental designs has been developed in order to reconcile the chromatographic conditions with the CL reaction. The method provides detection limits in the low microgl(-1) range and has been satisfactorily applied to the analysis of spiked raw milk samples.

Animals↗
PubMed2006

Release characteristics of salmon calcitonin from dextran hydrogels for colon-specific delivery.

Biodegradable dextran hydrogels were synthesized by crosslinking dextran (T-70) with epichlorohydrin (ECH) for the in vitro colon-specific delivery of salmon calcitonin (sCT). Crosslinking reaction was performed in 2.8 M NaOH solution both in the presence and absence of ethanol at 10 and 23 degrees C. Biodegradation kinetics of dextran hydrogels were studied and, in the presence of 0.7 IU ml-1 dextranase, dextran discs lost 71.0% and 56.5% of their dry weight within 80 h at pH 5.5 and 7.0, respectively. sCT was derivatized with the fluorescamine (FSM) at borate buffer (pH 9.0) and the quantitative determinations were performed using spectrofluorimetric method (lambdaex: 390 nm, lambdaem: 475 nm). In vitro release studies for the hydrogels prepared in the presence of ethanol were carried out in simulated gastrointestinal fluids. Results indicated that 84.9% of the loaded-sCT was released for 17 h and dextran hydrogel prepared in the presence of ethanol may be a good delivery device for the colon-specific delivery of other peptide-type drugs as well as sCT.

Animals↗
PubMed2004

Determination of milnacipran, a serotonin and noradrenaline reuptake inhibitor, in human plasma using liquid chromatography with spectrofluorimetric detection.

Milnacipran is an antidepressant drug belonging to the class of serotonin and noradrenaline reuptake inhibitors. A sensitive high performance liquid chromatographic during the development method coupled with a fluorimetric detection was set up, validated and then used routinely of the drug. After liquid-liquid extraction, milnacipran and its internal standard were analyzed by reversed-phase liquid chromatography (LC). The drug was derivatized with fluorescamine for fluorescence detection. The identity of the liquid chromatography peaks was controlled using mass spectrometry. The assay linearity was validated up to 1000 ng/ml. The limit of quantification was set at 5 ng/ml. Precision values (relative standard deviations) were lower than 5.4%, whereas the mean accuracy was higher than 95%. The extraction recoveries were higher than 70% for both milnacipran and the internal standard. In clinics, the LC-fluorescence method was routinely used to investigate the pharmacokinetics of milnacipran in patients and proved to be robust and capable of quantifying milnacipran in plasma for at least 36 h (four- to five-fold the elimination half-life).

Chromatography, Liquid↗
PubMed2005

One-step preparation of polyelectrolyte-coated PLGA microparticles and their functionalization with model ligands.

This work aimed at the development of a novel surfactant-free, one-step process for the concomitant formation of poly(lactide-co-glycolide) (PLGA) microparticles (MP) and surface coating with the polyelectrolyte chitosan, which is suitable for subsequent covalent conjugation of bioactive ligands. The technology is based on solvent extraction from an O/W-dispersion using a static micromixer. Surface coating occurred through interaction of the negatively charged, nascent PLGA MP with the polycationic chitosan, which was dissolved in the aqueous extraction fluid. Particles of 1-10 mum in diameter were produced with excellent reproducibility. The chitosan-coated PLGA MP were spherical and showed a smooth surface without pores, as demonstrated by scanning electron microscopy (SEM). The chitosan coatings were characterized by zeta potential measurements and X-ray photoelectron spectroscopy (XPS). The functional amino groups of chitosan were used to conjugate two model ligands to the coating, i.e. fluorescamine and NHS-PEG-biotin. The presence of the conjugated ligands was revealed by confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). Evidence for biotinylation was demonstrated through binding of fluorescently labelled streptavidin. The developed platform technology is straightforward and flexible. Future studies will focus on the design of microparticulate carriers with bioactive surfaces, e.g. as antigen delivery systems.

Biocompatible Materials↗
PubMed1977

High-performance liquid chromatographic assay of rat-brain dopamine and norepinephrine.

A high-performance liquid chromatographic method for the assay of dopamine (DM) and norepinephrine (NE) in rat brain has been developed. 3,4-Dihydroxybenzylamine was added to the sample as an internal standard. The isolated amines and the internal standard were subjected to reaction with fluorescamine, separated liquid chromatographically on a glycol-type gel and determined fluorimetrically. The standard deviation of the method was +/-2.7% for DM and +/-5.3% for NE. The contents of DM and NE in rat brain, with or without administration of a tyrosine hydroxylase inhibitor, as measured by the proposed method were always lower than those obtained by the trihydroxyindole method.

Animals↗
PubMed1982

Industrial hygiene air monitoring of phenylhydrazine.

Two gas chromatographic (GC) methods for the analysis of phenylhydrazine have been examined with phenyl- and rho-chlorophenylhydrazine and a new method using thin-layer chromatography (TLC) was developed. The NIOSH GC method (2-furalphenylhydrazone derivatization), which is classified as "proposed", was found to yield two derivatization peaks, which could only be separated with capillary columns. The other GC method (acetone phenylhydrazone derivatization) gave a single peak, however, of limited stability. In the newly developed TLC method the fluorescamine derivative of phenylhydrazine is separated and detected either visually or with a TLC-fluorescence scanner after fluorescence enhancement with paraffin wax. With ordinary TLC techniques the quantification of phenylhydrazine was possible down to 200 pg per spot. The sensitivity of the TLC method is generally sufficient for the requirements of industrial hygiene air monitoring of phenylhydrazine.

Air↗
PubMed1975

Determination of nanogram amounts of primary aromatic amines and nitro compounds in blood and plasma.

A rapid and highly sensitive method, based on the direct fluorimetric scanning of thin-layer chromatograms, is described for the quantitative determination of flunitrazepam and its main metabolites in human blood or plasma. This method is generally applicable to aromatic nitro compounds that are reducible with tin(II) chloride. In particular, it is possible to determine primary amines in nanogram amounts. Fluorescamine is used as a regent to produce fluorescent derivatives. The method is suitable for pharmacokinetic studies of flunitrazepam and its main metabolites in human blood and plasma.

Amines↗
PubMed1998

New, sensitive high-performance liquid chromatography method for the determination of slaframine in plasma and milk.

Slaframine was derivatized precolumn with fluorescamine. The derivatized slaframine was chromatographed isocratically using HPLC on a Hamilton PRP-1 C18 polymeric column with fluorescence detection. By using fluorescent derivatization, sensitivity was increased 100-fold over previously reported GC methods. A liquid-liquid partition was used to extract slaframine from plasma with a 95% recovery and a CV% of 8.4. A solid-phase extraction was used to extract slaframine from milk with a 91% recovery and a CV% of 9.8.

Alkaloids↗
PubMed1998

Determination of amoxicillin in human serum and plasma by high-performance liquid chromatography and on-line postcolumn derivatisation.

A high selective, sensitive and fast HPLC method developed for the determination in human serum and plasma. After protein precipitation with perchloric acid, an aliquot of the supernatant was neutralized by mixing it with sodium acetate solution and injected into a C18 HPLC column. Detection was done by a fluorescence detector after on-line postcolumn derivatisation with fluorescamine. The practical limit of quantification was 0.1 microgram/ml using 0.3 ml of plasma. Linearity was given in the tested range of 0.1 to 15 micrograms/ml plasma. Inter-day precision (relative standard deviation) over 7 days for 0.42 micrograms/ml was +/- 7.27%; for 4.54 micrograms/ml, +/- 5.24% and for 13.18 micrograms/ml, +/- 5.25%. Stability over 50 days in serum and plasma occurs at -70 degrees C but not at -20 degrees C (-25 to -35% reduction). This method was used for thousands of human serum and plasma samples.

Amoxicillin↗
PubMed1999

Determination of homotaurine as impurity in calcium acamprosate by capillary zone electrophoresis.

A method is reported which allows the quantification of homotaurine as an impurity in the drug. After addition of taurine as an internal standard, the sample is derivatised with fluorescamine at ambient temperature in 10 mM borate buffer, pH 9.2. The analytes are separated by capillary zone electrophoresis in a 31.2 cm (21 cm to the detector) x 100 microns I.D. fused-silica capillary at a potential of +7 kV and 25 degrees C. A 40 mM borate buffer, pH 9.2, is used as the electrolyte and detection is carried out at 205 nm. The validation tests showed that the method is reliable between 0.01% and 0.15% (m/m) of homotaurine with respect to the active drug. The limits of quantitation (0.01%, m/m) and detection (0.004%, m/m) allows to control the homotaurine content of the drug substance for which the maximum tolerated level is 0.05% (m/m). The proposed procedure (derivatisation and separation) developed in CE is rapid (20-25 min) by comparison to that currently used in HPLC (75 min). Satisfactory agreement was found between several batches of acamprosate analysed by CE and HPLC.

Acamprosate↗
PubMedDate not supplied

Determination of bone and tissue concentrations of teicoplanin mixed with hydroxyapatite cement to repair cortical defects.

A highly specific and sensitive isocratic reversed-phase high performance liquid chromatography (HPLC) method for the determination of the major component of teicoplanin in tissue is reported. Comparing fluorescamine and o-phthalaldehyde (OPA) as derivatizing agents, the derivative formed with the latter exhibits superior fluorescence intensity allowing detection of femtomole quantities. Pretreatment for tissue samples is by solid-phase extraction which uses Bakerbond PolarP C(18) cartridges and gives effective clean up from endogenous by-products. Linearity was given from 0.6 to 100 ng per injection. The coefficient of variation did not exceed 5.8% for both interday and intraday assays. It was found that when bone defects are repaired with a hydroxyapatite-teicoplanin mixture, the antibiotic does not degrade, even when it is in the cement for several months. The stability of teicoplanin in bone cement was determined fluorodensitometrically.

Animals↗
PubMed1982

Profiling of urinary medium-sized peptides in normal and uremic urine by high-performance liquid chromatography.

This report describes the profiling of medium-sized peptides in both normal and uremic urine by ion-pair reversed-phase high-performance liquid chromatography using an acetonitrile--heptafluorobutyric acid solvent system as eluent. Several medium-sized peptide peaks could be detected in both normal and uremic urine at low picomole level by using post-column fluorescence derivatization with fluorescamine. Contrary to expectation, uremic urine contained slightly larger amounts of medium-sized peptides compared with normal urine.

Chromatography, High Pressure Liquid↗