Biomedical subjects

H Irth

Publications and source records attributed to H Irth.

At least 37 records · Page 2Linked to original sources

PubMed2000

Free flow electrophoresis device for continuous on-line separation in analytical systems. An application in biochemical detection.

A free flow electrophoresis (FFE) device was developed for continuous electrophoretic separation of charged compounds and implemented in a continuous flow biochemical detection (BCD) system. These continuous separation characteristics make FFE well suitable for online implementation in a chromatographic or flow injection analysis system, in which an additional separation step of charged compounds is desired. In a heterogeneous biochemical flow assay for the determination of biotin, an analyte zone reacts with an excess of an affinity protein. Subsequently, the free binding sites of the affinity protein react with an excess of fluorescein-labeled ligand. Free and affinity protein-bound label are separated on the FFE device prior to fluorescence detection of the separated fractions. Biotin and streptavidin were chosen as, respectively, model ligand and affinity protein. Since all the compounds that are involved possess different electrophoretic properties, quantitative analysis is performed after completely separating the fluorescent affinity complex and labeled biotin in the FFE device within 2 min. Since the device is optically transparent, the separated zones can be detected in the separation compartment, using laser-induced fluorescence. The applicability of the BCD-FFE system in combination with a HPLC separation is demonstrated in the bioanalysis of biotin in human urine at the micromole per liter level.

Biotin↗

PubMed2000

Label-free biochemical detection coupled on-line to liquid chromatography

The on-line coupling of a label-free optical biosensor to a HPLC system is described by combining the separation power of HPLC with the specificity of the biosensor system. A highly cross-reactive antibody against the pesticide isoproturon was used as model for affinity proteins. The binding strength of the antibody to the utilized pesticides was characterized with the biosensor, first. In the on-line coupling setup, the eluate of the HPLC was mixed continuously with the antibodies. The presence of antigens was detected by a reduction of the antibody binding to the transducer. This reduced binding was quantified by a differentiation of the sensor signal by applying a Savitzky-Golay algorithm. Limits of detection were found to be in the femtomole range without preconcentration, which is comparable to a study using fluorescence-based biochemical detection.

Journal Article↗

PubMed2000

High-performance liquid chromatography coupled to enzyme-amplified biochemical detection for the analysis of hemoglobin after pre-column biotinylation.

The determination of proteins with enzyme-amplified biochemical detection (EA-BCD) coupled on-line with high-performance liquid chromatography (HPLC) is demonstrated. The EA-BCD system was developed to detect biotin-containing compounds. Hemoglobin, which was used as a model compound, was biotinylated prior to sample introduction. Several biotinylation parameters, such as pH and removal of excess biotinylation reagent, were investigated. After biotinylation samples were introduced to HPLC followed by EA-BCD. To the HPLC effluent, alkaline phosphatase label streptavidin (S-AP) was added, which possesses high affinity to biotin and biotin-containing compounds. Excess S-AP was removed by means of an immobilized biotin column followed by substrate addition. The non-fluorescent substrate is converted to a highly fluorescent product by the enzyme label. A detection limit of 2 femtomol biotinylated Hb was achieved with good reproducibility and linearity. However, biotinylation at low analyte concentration suffers from low yield due to slow reaction kinetics. Finally, Hb was successfully extracted from urine with a recovery of 94%.

Alkaline Phosphatase↗

PubMed2000

High-performance liquid chromatography with on-line coupled UV, mass spectrometric and biochemical detection for identification of acetylcholinesterase inhibitors from natural products.

A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.

Acetylcholinesterase↗

PubMed2000

The application of HPLC with on-line coupled UV/MS-biochemical detection for isolation of an acetylcholinesterase inhibitor from narcissus 'Sir Winston Churchill'.

An HPLC with on-line coupled UV/MS-biochemical detection method for acetylcholinesterase (AChE) inhibitors in natural sources has been developed. The potential of this method is shown by the isolation of a new AChE inhibitor from the alcoholic extract of Narcissus 'Sir Winston Churchill'. Combining a prefractionation technique using centrifugal partition chromatography with the on-line HPLC-UV/MS-biochemical detection resulted in the isolation of the active compound that was identified as ungiminorine. This alkaloid shows a mild inhibitory effect on AChE.

Alkaloids↗

PubMed1999

Enzyme amplification as detection tool in continuous-flow systems. I. Development of an enzyme-amplified biochemical detection system coupled on-line to flow-injection analysis.

The on-line coupling of flow-injection analysis (FIA) to an enzyme-amplified biochemical detection (EA-BCD) system is described. The aim of this study is the development of a detection system able to detect biotin-containing compounds at low concentration levels. The detection system is based on the interaction of biotin with enzyme-labeled affinity proteins. Biotin possesses a high affinity to both streptavidin and anti-biotin Fab fragments, which are both tested. Several biotin derivatives are available with different reactive probes, which can be used to label analytes of interest. Therefore, biotin acts as a universal probe for the enzyme-amplified biochemical detection. Alkaline phosphatase (AP) was used as enzyme label. Several parameters, such as substrate type and concentration, concentration of enzyme-labeled affinity protein, reaction time and reaction temperature were examined. Biotin aminocaproic acid was used as a model compound. In addition to biotin aminocaproic hydrazide, other biotinylation reagents were also examined. With fluorescence detection of the enzyme-generated product, a mass detection limit of 1 fmol was achieved.

Biotin↗

PubMed1999

Enzyme amplification as detection tool in continuous-flow systems. II. On-line coupling of liquid chromatography to enzyme-amplified biochemical detection after pre-column derivatization with biotin.

Enzyme-amplified biochemical detection (EA-BCD) was used as a post-column detection technique, coupled on-line with high-performance liquid chromatography (HPLC). The enzyme detection system was developed to detect biotin or biotin containing compounds. Biotinylation is widely used to label analytes of interest ranging from small molecules to proteins and DNA. Naphthalene aldehyde and anthracene aldehyde were used as model compounds. Both compounds were biotinylated off-line with biotin aminocaproic hydrazide (BACH). On-column biotinylation was performed by preconcentration of anthracene aldehyde on copper phthalocyanine. After biotinylation, samples were introduced to the HPLC system. Enzyme-labeled streptavidin, which possesses high affinity to biotin, was added post-column to the HPLC effluent. Excess of enzyme-labeled affinity protein was removed by means of an immobilized biotin column. After separation of free and bound fraction, substrate was added, which was converted to a fluorescent product by the enzyme label. Using alkaline phosphatase as an enzyme label, a mass detection limit after on-column preconcentration and biotinylation of 250 fmol was achieved.

Biotin↗

PubMed1998

Gradient reversed-phase liquid chromatography coupled on-line to receptor-affinity detection based on the urokinase receptor.

A postcolumn receptor-affinity detection (RAD) was developed for the detection of urokinase and cross-reactive compounds. The analytical method consisted of gradient reversed-phase HPLC coupled on-line to a RAD system based on fluorescein-labelled urokinase receptor (fluorescein-uPAR) as reagent. Fluorescein-uPAR was added continuously to the HPLC effluent to react with analytes eluting from the LC column. Unreacted fluorescein-uPAR was removed by a short affinity column packed with an immobilised urokinase support. The analyte-bound fluorescein-uPAR fraction passes the affinity column unretained and was detected downstream by means of a fluorescence detector. An absolute detection limit of 40 fmol urokinase was obtained in the flow injection mode. In the gradient HPLC-RAD system a detection limit of 40 nM (20-microl injection, absolute amount, 800 fmol) was obtained. The present method allowed the identification of active breakdown products of urokinase both in standard samples and biological matrices.

Chromatography, Affinity↗

PubMed1998

Optimisation of a heterogeneous non-competitive flow immunoassay comparing fluorescein, peroxidase and alkaline phosphatase as labels.

Off- and on-line strategies for a non-competitive heterogeneous flow immunoassay were developed comparing three different labels. The samples, containing the model compounds digoxin or digoxigenin, were either pre-incubated off-line or on-line in a mixing coil with excess of labelled anti-digoxigenin Fab-fragments. The excess of Fab-fragments was then separated from the digoxin bound Fab-fragments by passing the sample through a column with immobilised digoxin. The off-line immunochemical detection system is suitable for sensitive high through-put screening of the analytes, whereas the on-line system is more suitable for coupling as a post-column detection unit to liquid chromatography. The digoxin and digoxigenin content in the sample were quantified using fluorescein (F) and enzyme (peroxidase (POD), alkaline phosphatase (AP)) labelled Fab-fragments. The fluorescein label was directly measured with the fluorescence detector, whereas a fluorescent enzyme product was measured in the two enzyme based systems, using 3-(p-hydroxyphenyl)-propionic acid (HPPA) and hydrogen peroxide for POD and, and 4-methylumbelliferyl phosphate (4-MUP) for AP. The highest sensitivity and lowest limit of detection (LOD) was obtained with the Fab-POD system with LODs for digoxin and digoxigenin in the off- and on-line configurations of 0.025 and 0.01 nM, respectively. The sample through-put for the off- and on-line systems were 43 and 32 samples per hour, respectively.

Alkaline Phosphatase↗

PubMed1997

Theoretical concepts of on-line liquid chromatographic-biochemical detection systems. I. Detection systems based on labelled ligands.

A theoretical foundation for on-line coupling of liquid chromatography (LC) with fluororeceptor assays based on fluorescent ligands is presented. Using a recently developed LC-receptor affinity detection (RAD) system as a model, equations are derived which describe the detector response and the signal-to-noise ratio as a function of important biochemical and instrumental parameters. The effect of ligand and label affinity, and receptor concentrations on the detector performance were investigated. It was found that the response of the RAD system is correlated with the affinity of the injected compounds and that the relative affinity order of binding ligands is maintained in the RAD system.

Biological Assay↗

PubMed1997

Theoretical concepts of on-line liquid chromatographic-biochemical detection systems. II. Detection systems based on labelled affinity proteins.

A theoretical concept for on-line liquid chromatography-biochemical detection (LC-BCD) using labelled affinity proteins as reporter molecules is presented. The BCD system is based on the post-column addition of labelled affinity proteins such as fluorescein-labelled streptavidin to the LC effluent. After a short reaction time, free and analyte-bound label are separated during passage through a column packed with an immobilised-ligand support. The bound fraction passes the column unretained and is measured downstream by means of a conventional HPLC detector. The theoretical model presented here relates the detector response to the most important instrumental and biochemical parameters such as dispersion, reaction time, concentration and affinity of the affinity protein and the number of binding sites. The theoretical concept is validated using fluorescein-labelled streptavidin and biotin as model system.

Binding Sites↗

PubMed1997

Liquid chromatography-mass spectrometry with on-line solid-phase extraction by a restricted-access C18 precolumn for direct plasma and urine injection.

In this paper, the on-line coupling of solid-phase extraction, based on a restricted-access support with liquid chromatography-mass spectrometry (LC-MS), for the analysis of biological samples is described. The system was tested with cortisol and prednisolone for plasma analysis and arachidonic acid for urine analysis. A precolumn packed with a 25-micron C18 alkyl-diol support is used for direct plasma or urine injection. Using column-switching techniques, the analytes enriched on the precolumn are eluted to the analytical column without transfer loss. An on-line heart-cut technique was employed and only the analyte-containing fraction eluting from the LC column is directed to the MS to protect the LC-MS interface and ion-source from contamination. The whole system is operated in a parallel mode, that is, sample pre-treatment and LC-MS analysis are performed simultaneously to provide the shortest possible analysis time. The only off-line sample pre-treatment step required was centrifugation to remove particulate matter. With the fully automated system, total analysis times of 5 and 9.5 min were achieved for cortisol in serum and arachidonic acid in urine, respectively. Cortisol and related compounds were quantitatively recovered from plasma with a detection limit for prednisolone (direct injection of 100 microliters on restricted-access precolumn) of 2 ng/ml.

Arachidonic Acid↗

PubMed1996

Applying hollow fibres for separating free and bound label in continuous-flow immunochemical detection.

On-line liquid chromatography-immunochemical detection (LC-ICD) provides the possibility to individually monitor cross-reactive compounds overcoming the need of tedious fraction collection. ICD is performed as a post-column reaction detection system and is based on a two-step immunoreaction. In the first step unlabelled antibodies are added to the LC effluent and allowed to react with antigens (analytes) eluting from the LC column. The amount of analytes bound to the antibodies is measured by adding, in a second step, labelled antigen to the reaction mixture. For quantitation, free and bound label need to be separated prior to detection. The present paper describes a hollow fibre module (HFM), which can be used for this purpose. Separation of free and bound label occurs on discrimination by size. Using biotin as a model compound, a detection limit of 30 nmol/l can be reached employing anti-biotin antibodies and a low-molecular-mass fluorescence label in the LC-ICD system. Additional to low-molecular-mass labels, the HFM allows the use of small enzyme labels. In this context, horseradish peroxidase-labelled biotin was used as a label in combination with antibodies in the immunochemical detection of biotin. This allows future implementation of commercially available enzyme immunoassay kits in continuous-flow immunochemical detection.

Antigens↗

PubMed1996

Simultaneous determination of cross-reactive leukotrienes in biological matrices using on-line liquid chromatography immunochemical detection.

Sulfidopeptide leukotrienes, which are important biomarkers for several diseases, are commonly measured by microtiter plate immunoassays. These immunoassays, however, cannot distinguish between several structurally similar leukotrienes and their cross-reactive metabolites and, therefore, need extensive sample handling and fractionation by means of liquid chromatography (LC). This paper describes the development and automation of a continuous-flow immunochemical detection (ICD) system and its subsequent on-line coupling to LC. The on-line LC-ICD system based on fluorescence-labeled leukotriene E4 (LTE4) was used to determine sulfidopeptide leukotrienes and their cross-reactive metabolites in a single run. Furthermore, biological matrices, e.g., urine and human cell extracts, were analyzed, the only sample pretreatment being on-line solid-phase extraction (SPE) on a novel RP-C4 restricted-access support. The determination limit of LTE4 in urine was 0.2 ng/mL (800 fmol; injection volume, 2000 microL; signal-to-noise ratio, 10). The system was linear from 0.2 to 1.0 ng/mL LTE4. Using nonlinear curve-fitting, the range could be expanded to 2.5 ng/mL. It is shown that, besides quantitation of known analytes, on-line LC-ICD is useful in the discovery of cross-reactive LTE4 metabolites.

Antigen-Antibody Reactions↗

PubMed1996

Reversed-phase liquid chromatography coupled on-line to receptor affinity detection based on the human estrogen receptor.

On-line coupling of reversed-phase liquid chromatography to a biochemical detection system based on receptor-ligand interactions is described. The receptor affinity detection is performed using a postcolumn reaction detection system with open-tubular reaction coils. In the first step, a solution containing the steroid binding domain of the human estrogen receptor is added to the LC effluent via a mixing union, and the mixture is allowed to react. In the second step, a fluorescent estrogenic ligand, coumestrol, is added to saturate the remaining free binding sites of the estrogen receptor. Both heterogeneous and homogeneous detection systems have been developed. In the former case, free and receptor-bound coumestrol were separated prior to detection by means of a short column containing a restricted-access reversed-phase support. Since free and receptor-bound coumestrol differ significantly in fluorescence intensity, the system can also be operated in the homogeneous mode without requiring a separation step. Using 5 nmol/L of human estrogen receptor, a detection limit of 5 nmol/L was obtained for compounds possessing a high affinity for the estrogen receptor, such as 17 beta-estradiol and diethylstilbestrol. Detection limits for weaker ligands amount to 20 nmol/L. Non-estrogenic steroids such as methyltestosterone or progesterone are not detected at all. The selectivity of the present method is demonstrated for the analysis of urine samples.

Chromatography, Affinity↗

PubMed1995

Determination of 1,2,6-inositol trisphosphate (derivatives) in plasma using iron(III)-loaded adsorbents and capillary zone electrophoresis-(indirect) UV detection.

A method for the determination of 1,2,6-inositol trisphosphate (IP3) and derivatives in plasma by capillary zone electrophoresis with (indirect) UV detection has been developed. The sample pretreatment is based on the selective isolation after complexation of inositol phosphates with iron(III) loaded on an adsorbent. Plasma protein denaturation was performed with sodium dodecyl sulfate. The selectivity of the method is demonstrated with the analysis of phenylacetate-IP3. The recoveries amount to 65% and 88% in plasma and in water, respectively.

Adsorption↗

PubMed1994

Liquid chromatographic determination of the adenosine receptor agonist CGS 21680 in blood using on-line solid-phase extraction on a phenylboronic acid support and fluorescence detection.

An analytical method is described for the selective determination of A1 or A2 adenosine receptor agonists in blood. By implementing solid-phase extraction using immobilized-phenylboronic acid (PBA) in sample pretreatment, all adenosine derivatives are retained via their intact cis-diol group. On-line desorption of the analytes from the PBA support to the C18 analytical column is performed by injection of a small plug of perchloric acid. Fluorescence and UV detection are employed for the different adenosine derivatives. The method is applied to the determination of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N- ethylcarboxyamidoadenosine (CGS 21680, I) in blood using fluorescence detection. The only off-line sample handling step is the extraction of blood with ethyl acetate and subsequent evaporation of the extraction solvent. The detection limit of the method was 0.25 ng (signal-to-noise ratio 3:1) and the determination limit for I in blood (pretreatment of 100 microliters) was 5 ng/ml. The method was validated and used to study the pharmacokinetics of I in rats.

Acetates↗

PubMed1994

Potential of receptor-ligand interactions for sample handling in liquid and gas chromatography.

The use of receptor proteins for biospecific sample handling in liquid and gas chromatography is described. As a model system the uterine estrogen receptor was chosen for the isolation of 17 beta-estradiol and its synthetic agonist diethylstilbestrol. Biochemical characteristics relevant for the use of receptors in sample handling such as the kinetics of receptor-ligand binding, reproducibility and capacity are examined by means of an estrogen radioreceptor assay. Different techniques for the non-covalent immobilization of the estrogen receptor were investigated. Both protamine-coated glass fibre filters and silica particles which bind the receptor via electrostatic interactions have been used for this purpose. The use of the estrogen receptor in the isolation of 17 beta-estradiol and diethylstilbestrol prior to GC-MS analysis is demonstrated and discussed.

Animals↗