Biomedical subjects

Constantinos K Zacharis

Publications and source records attributed to Constantinos K Zacharis.

7 recordsLinked to original sources

PubMed2006

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

Animals↗

PubMed2006

Automated sample treatment by flow techniques prior to liquid-phase separations.

Sample preparation (SP) is an integral and important part of an analytical process. Lately, SP has been the topic of increased interest in research and development of novel advanced technologies. Major needs in this aspect are miniaturization, automation, and enrichment. Among other methodologies, flow techniques can be conveniently and effectively coupled to liquid-phase separation technologies for on-line sample preparation. This paper reviews the current trends in on-line automated sample preparation by flow-through techniques prior to liquid-phase separations. Strategies and interfaces developed to couple flow techniques with liquid chromatography, capillary electrophoresis, and mass spectrometry are described. Advantages and limitations of the coupling are discussed. The paper also highlights certain selected applications of these coupled systems.

Chromatography, Liquid↗

PubMed2006

Coupling of sequential injection analysis and capillary electrophoresis - Laser-induced fluorescence via a valve interface for on-line derivatization and analysis of amino acids and peptides.

The on-line coupling of sequential injection analysis (SIA) and capillary electrophoresis (CE) via an in-line injection valve is presented. The SIA system is used for automated derivatization of amino acids and peptides. Dichlorotriazinylaminofluorescein serves as the derivatization agent, thus enabling sensitive laser-induced fluorescence detection of the derivatized analytes. The SIA procedure includes the following steps: (a) introduction of reagent and sample zones in a holding coil, (b) sample and reagent mixing in a reaction coil, (c) stop-flow step for increase of the reaction time, and (d) delivery of derivatized sample into the loop of the micro-valve interface. A small portion of the analyte zone is introduced electrokinetically in the separation capillary via the valve interface and CE analysis is performed. Factors affecting the CE separation, such as pH, the borate and sodium dodecyl sulphate concentration of the background electrolyte have been optimized. The derivatization conditions have been studied to obtain a high reaction yield in a relative short time. The transfer of a part of the reaction plug into the loop of the valve interface has been optimized. Using des-Tyr(1)-[Met]-enkephalinamide as test compound, it is demonstrated that after automated derivatization, on-line electrophoretic analysis could be achieved. Glycine has been selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For the enkephalin, good reproducibility (RSD<4.5% calculated by the ratio of the peak areas) and linearity (0.5-5 microg mL(-1), R(2)>or=0.994) are obtained with a detection limit of 30 ng mL(-1) (S/N=3).

Amino Acids↗

PubMed2006

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies.

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.

Anion Exchange Resins↗

PubMed2004

On-line coupling of sequential injection with liquid chromatography for the automated derivatization and determination of gamma-aminobutyric acid in human biological fluids.

The principle of sequential injection analysis (SIA) was exploited to develop a rapid fully automated and efficient pre-column derivatization procedure coupled on-line to liquid chromatography (HPLC). Using the SIA-HPLC derivatization protocol gamma-aminobutyric acid (GABA) was determined fluorimetrically in human biological fluids with o-phthaldialdehyde (OPA) as derivatization reagent and minimum sample pretreatment. A lab-built SIA system was used to handle samples, standard solutions and OPA reagent. Appropriate volumes of the reagents were introduced in the holding coil of the SIA system and were mixed on propulsion to the HPLC loop through a suitable reaction coil. The chemical (pH, c(OPA), c(mercaptoethanol)) and instrumental variables (volumes of sample and reagent, reaction time) of the reaction were studied and optimized in terms of maximum sensitivity. The chromatographic variables (gradient composition of the eluent and flow rate) were studied for optimum selectivity and peak characteristics. The developed experimental configuration facilitated fully-automated operation thus minimizing errors in handling. Additionally the method as a whole provided very satisfactory sensitivity, precision and accuracy. Direct determination of GABA in human urine and cerebrospinal fluid (CSF) at microg L(-1) (ppb) levels was accomplished, with minimum sample pretreatment.

Automation↗

PubMed2004

Automated sample preparation based on the sequential injection principle. Solid-phase extraction on a molecularly imprinted polymer coupled on-line to high-performance liquid chromatography.

A molecularly imprinted polymer (MIP) prepared using caffeine, as a template, was validated as a selective sorbent for solid-phase extraction (SPE), within an automated on-line sample preparation method. The polymer produced was packed in a polypropylene cartridge, which was incorporated in a flow system prior to the HPLC analytical instrumentation. The principle of sequential injection was utilised for a rapid automated and efficient SPE procedure on the MIP. Samples, buffers, washing and elution solvents were introduced to the extraction cartridge via a peristaltic pump and a multi-position valve, both controlled by appropriate software developed in-house. The method was optimised in terms of flow rates, extraction time and volume. After extraction, the final eluent from the extraction cartridge was directed to the injection loop and was subsequently analysed on HPLC. The overall set-up facilitated unattended operation, operation and improved both mixing fluidics and method development flexibility. This system may be readily built in the laboratory and can be further used as an automated platform for on-line sample preparation.

Automation↗

PubMed2004

Rapid spectrofluorimetric determination of lisinopril in pharmaceutical tablets using sequential injection analysis.

The present work reports for the first time a simple and rapid method for the spectrofluorimetric determination of lisinopril (LSP) in pharmaceutical formulations using sequential injection analysis (SIA). The method is based on reaction of LSP with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol (borate buffer medium, pH=10.6). The emission of the derivative is monitored at 455 nm upon excitation at 346 nm. The various chemical and physical conditions that affected the reaction were studied. The calibration curve was linear in the range 0.3-10.0 mg L(-1) LSP, at a sampling rate of 60 injections h(-1). Consumption of OPA reagent was significantly reduced compared with conventional flow injection (FI) systems, because only 50 microL of OPA was consumed per run. The method was found to be adequately precise ( s(r)=2% at 5 mg L(-1) LSP, n=10) and the 3 sigma detection limit was 0.1 mg L(-1). The method was successfully applied to the analysis of two pharmaceutical formulations containing LSP. The results obtained were in good agreement with those obtained by use of high-performance liquid chromatography (HPLC), because the mean relative error, e(r), was <1.8%.

Calibration↗