Biomedical subjects

W Thormann

Publications and source records attributed to W Thormann.

At least 73 records · Page 4Linked to original sources

PubMed1995

Modification of a tunable UV-visible capillary electrophoresis detector for simultaneous absorbance and fluorescence detection: profiling of body fluids for drugs and endogenous compounds.

Using fused-silica optical fibres for fluorescence light collection and bandpass filters for selection of emission wavelengths, a capillary electrophoresis detection cell of a conventional, tunable UV-Vis absorbance detector was adapted for simultaneous fluorescence (at selected emission wavelength) and absorbance (at selected excitation wavelength) detection. Detector performance is demonstrated with the monitoring of underivatized fluorescent compounds in body fluids by micellar electrokinetic capillary chromatography with direct sample injection. Compared with UV absorption detection, fluorescence detection is shown to provide increased selectivity and for selected compounds also up to tenfold higher sensitivity. Examples studied include screening for urinary indole derivatives (tryptophan, 5-hydroxytryptophan, tyrosine, 3-indoxyl sulfate and 5-hydroxyindole-3-acetic acid) and catecholamine metabolites (homovanillic acid and vanillylmandelic acid) and the monitoring of naproxen in serum, quinidine in serum and urine and of salicylate and its metabolites in serum and urine.

Catecholamines↗

PubMed1995

Exploration of the metabolism of dihydrocodeine via determination of its metabolites in human urine using micellar electrokinetic capillary chromatography.

After single-dose administration of 40 or 60 mg of dihydrocodeine (DHC, in a slow-release tablet) to four healthy individuals known to be extensive metabolizers of debrisoquine, the urinary excretion of DHC and its four major metabolites, dihydrocodeine-6-glucuronide, nordihydrocodeine, dihydromorphine and nordihydromorphine, was assessed using micellar electrokinetic capillary chromatography (MECC). DHC and two of its metabolites (dihydrocodeine-6-glucuronide and nordihydrocodeine) could be analyzed by direct urine injection, whereas the metabolic pattern was obtained by copolymeric bonded-phase extraction of the solutes from both plain and hydrolyzed urine specimens prior to analysis. The total DHC equivalents excreted within 8 and 24 h were determined to be 30.4 +/- 7.7% (n = 5) and 63.8 +/- 6.1% (n = 2), respectively, and only about 4% of the excreted DHC equivalents were identified as morphinoids. Furthermore, almost no morphinoid metabolites of DHC could be found after administration of quinidine (200 mg of quinidine sulfate) 2 h prior to DHC intake.

Chromatography, Liquid↗

PubMed1995

Feasibility study of a drug immunoassay based on micellar electrokinetic capillary chromatography with laser induced fluorescence detection: determination of theophylline in serum.

This paper presents principle and first results of a novel competitive binding immunoassay for monitoring of theophylline in human serum. The assay is based upon short time incubation of a mixture of antiserum, containing the antibody raised against theophylline, fluorescein labelled theophylline (tracer) and serum prior to injection of a few nanoliter of this mixture onto a fused-silica capillary for subsequent separation and analysis of free tracer and the antibody-tracer-complex by micellar electrokinetic capillary chromatography with laser induced fluorescence detection. Quantitation based upon multi-level calibration using the height of the peak produced by the free tracer is shown to provide theophylline serum levels which are in agreement with those obtained by a commercial fluorescence polarization immunoassay and with those determined by micellar elektrokinetic capillary chromatography with direct serum injection and on-column UV absorption detection.

Binding, Competitive↗

PubMed1995

Impact of electroosmosis on isotachophoresis in open-tubular fused-silica capillaries: analysis of the evolution of a stationary steady-state zone structure by computer simulation and experimental validation.

A dynamic computer model for simulation of open-tubular capillary electrophoresis that includes in situ calculation of electroosmosis along the fused-silica capillary column has been applied to the characterization of an anionic isotachophoretic system in presence of a cathodic electroosmotic flow. For each column segment, electroosmosis is calculated with the use of a wall mobility, the voltage gradient and the degree of dissociation of the silanol surface groups of the capillary wall. Then, the bulk capillary flow is taken to be the average of all of the segment flows and considered to represent a plug flow. This simple approach enables the combined simulation of the temporal behavior of an isotachophoretic zone structure in presence of electroosmosis. For a model anionic isotachophoretic configuration at pH 6, simulation data reveal the asymptotic formation of a stationary steady-state zone configuration in which electrophoretic and electroosmotic zone displacements are opposite and of equal magnitude. The position of the stationary boundaries are predicted to be dependent on the selected wall pK and mobility values. For two different instruments, qualitative agreement between experimental data and simulation results obtained with a wall pK between 5 and 6 is demonstrated. However, for the two experimental setups, significant differences in electroosmotic pumping (i.e. wall mobility values) are noted.

Anions↗

PubMed1995

Sample self-stacking and sample stacking in zone electrophoresis with major sample components of like charge: general model and scheme of possible modes.

A theoretical study is presented of zone electrophoretic behavior of samples that contain one or more minor analytes and at least one major ionogenic component of like charge. Based on a simple model comprising weak univalent anionic electrolytes, conditions are derived under which analytes are temporarily focused isotachophoretically into very narrow zones by a sample self-stacking effect provided by the major sample components. Requirements for minimal/maximal mobility and a background coion concentration dependent minimal concentration of a major sample component (stacker) are presented. For systems in which sample self-stacking does not apply, an expression for the concentrating factor is derived that involves the effects of both nonselective (classical) and selective sample stacking, the latter being a consequence of electrophoretic separation of the minor analyte from the major component. The theory derived is discussed with selected model examples by using both numerical calculation and computer simulation.

Electric Conductivity↗

PubMed1994

Determination of methadone and its primary metabolite in human urine by capillary electrophoretic techniques.

Data presented in this paper show that methadone and its primary urinary metabolite (M1) can not easily be determined by SDS-based micellar electrokinetic chromatography; however, they separate rapidly under cationic capillary zone electrophoretic conditions using a borate buffer with a pH of ca. 9. Eight urines obtained from individuals undergoing methadone therapy, which tested markedly positive for methadone using an enzyme multiplied immunoassay and in which the presence of methadone and M1 was also confirmed by GC-MS, have been analyzed. Using an extraction procedure with disposable cartridges containing a copolymeric sorbent, the presence of methadone and M1 could be confirmed in all urines, whereas with direct urine injection, the two compounds could only be determined in six urines. Thus, for unambiguous confirmation by capillary electrophoresis, extraction of the compounds of interest is preferred. The described assay is rapid (with typical run times being less than 6 min), free of interferences from coextracting drugs of abuse and/or their major metabolites, and characterized by a good reproducibility. After extraction of 5 ml urine, drug concentrations down to ca. 20 ng/ml can be monitored unambiguously.

Biotransformation↗

PubMed1994

Screening for hydroxylation and acetylation polymorphisms in man via simultaneous analysis of urinary metabolites of mephenytoin, dextromethorphan and caffeine by capillary electrophoretic procedures.

Phenotypes for hydroxylation can be predicted by using mephenytoin and dextromethorphan as substrates, whereas phenotypes for acetylation can be determined with caffeine as probe drug. After single-dose administration of one of these drugs, of two of them simultaneously, or of the three drugs together, the major urinary metabolites (4-hydroxymephenytoin; dextrorphan, 3-methoxymorphinan, 3-hydroxymorphinan; 5-acetylamino-6-amino-3-methyluracil as decomposition product of 5-acetylamino-6-formylamino-3-methyluracil, 1-methylxanthine, respectively) of these substrates were analyzed by capillary electrophoretic techniques. No sample pretreatment other than enzymatic hydrolysis of the conjugated compounds was applied. Assays based on micellar electrokinetic capillary chromatography are shown to allow simultaneous and unambiguous phenotyping with mephenytoin and dextromethorphan or mephenytoin and caffeine. Simultaneous screening for all three polymorphisms with a single injection of a hydrolyzed urine is shown to be possible via use of multiwavelength absorption detection only. Phenotypes determined by electrokinetic capillary techniques are shown to agree with those obtained by analysis with customary assays based on high-performance liquid chromatography.

Acetylation↗

PubMed1994

Clinical and forensic applications of capillary electrophoresis.

This survey is aimed at giving the readers a short overview of the present state of the art of clinical and forensic applications of capillary electrophoresis. First, the principles associated with electrokinetic capillary separations and instrumentation, sample preparation and solute quantitation are briefly discussed. This is followed by chapters describing the determination of endogenous and exogenous compounds in body fluids and tissue extracts. Finally, a survey of major achievements including reference to fully developed electrokinetic capillary assays is provided. The paper concludes with a brief outlook.

Body Fluids↗

PubMed1994

Application of dynamic capillary isoelectric focusing to the analysis of human hemoglobin variants.

Capillary isoelectric focusing (CIEF) with electro-osmotic zone displacement of normal and pathological hemoglobins (Hb) is reported. CIEF is performed in untreated, open-tubular, fused-silica capillaries of 75 microns internal diameter using methylcellulose for dynamic column conditioning. After direct injection of hemolysates mixed with carrier ampholytes, high resolution separation of Hb variants, including Hb A1c, A, F, D, S, E and A2, is obtained, this permitting unambiguous characterization of Hb patterns of normal adults, newborns, patients with diabetes, different hemoglobinopathies and thalassemia syndromes. Qualitatively, the CIEF data compare well with those obtained by gel isoelectric focusing and high-performance liquid chromatography. CIEF is demonstrated to be a simple, rapid and fully instrumental approach to Hb analysis. Run times of less than 20 min make CIEF an attractive method for routine Hb investigations and screening programs.

Adult↗

PubMed1994

Factors affecting the determination of drugs and endogenous low molecular mass compounds in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

Factors influencing the establishment of an analytical window in front of the solubilized proteins in micellar electrokinetic capillary chromatography (MECC) with direct serum injection (DSI) are discussed. Both drugs and endogenous low molecular mass compounds eluting within the analytical window are identified concurrently by multi-wavelength absorption detection. Variables such as the concentration of the micelle forming substance, ionic strength, applied voltage, initial sample zone length, capillary length, selected buffer additives, insufficient renewal of the buffer in the anodic buffer vial and sample matrix are shown to impact MECC of endogenous compounds and model drugs, such as antiepileptics. For two drugs eluting within the analytical window, phenobarbital and ethosuximide, serum levels determined by DSI with external calibration are shown to compare well with levels obtained after liquid-liquid extraction and internal calibration (use of an internal standard). In addition, reproducibility of both assays is excellent. The limit of employing DSI is demonstrated with the determination of the hydrophobic drug phenytoin. Using an automated, commercial instrument and naproxen as model drug, high-speed MECC separations of high reproducibility and with a throughput of 12-15 samples per h are presented.

Anticonvulsants↗

PubMed1994

Micellar electrokinetic capillary chromatography of benzodiazepines in human urine.

The determination of the major urinary compounds of eight common benzodiazepines, flunitrazepam, diazepam, midazolam, clonazepam, bromazepam, temazepam, oxazepam, and lorazepam, by micellar electrokinetic capillary chromatography (MECC) is shown to be a simple and attractive approach for confirmation testing of these drugs in human urine. After enzymatic hydrolysis and extraction using mixed-mode solid-phase cartridges and a two-step elution protocol, fractions were analyzed in a phosphate/borate buffer (pH 9.3) containing 75 mM sodium dodecyl sulfate and small amounts of isopropanol, methanol and/or acetonitrile using an instrument with on- column multi-wavelength detection. The presence of these compounds could unambiguously be confirmed in patient urines which tested positive for benzodiazepines using a commercial enzyme multiplied immunoassay screening technique (EMIT). The sensitivity of the MECC assay is demonstrated to be better than that of EMIT. MECC analysis of one patient urine which tested negatively employing EMIT revealed the presence of lorazepam, this demonstrating that false-negative results from the initial immunological screening process can be recognized using MECC. For one example, 7-aminoflunitrazepam, the MECC data are shown to agree well with those obtained by gas chromatography/mass spectrometry.

Benzodiazepines↗

PubMed1994

Analysis of mephenytoin, 4-hydroxymephenytoin and 4-hydroxyphenytoin enantiomers in human urine by cyclodextrin micellar electrokinetic capillary chromatography: simple determination of a hydroxylation polymorphism in man.

Using cyclodextrin micellar electrokinetic capillary chromatography (CD-MECC), baseline separation of mephenytoin, 4-hydroxymephenytoin and 4-hydroxyphenytoin enantiomers in urine was effected with beta-cyclodextrin. After single-dose administration of 100 mg of racemic mephenytoin, the 0-8 h urine was collected, and enzymatically hydrolyzed urine specimens were applied. For extensive metabolizers, a single peak for 4-hydroxymephenytoin was detected corresponding to the S-enantiomer. This peak was either very small or undetectable in samples of poor metabolizers. Typically, mephenytoin could not be detected in these samples. However, application of undeglucuronidated extracts revealed the presence of free S-4-hydroxymephenytoin and R,S-mephenytoin and thus permitted phenotyping via both the urinary S:R enantiomeric ratio of mephenytoin and the hydroxylated metabolite. Application of enzymatically hydrolyzed and extracted urines after phenytoin administration (100 mg; 0-8 h urine collection) revealed the presence of S-4-hydroxyphenytoin. Thus, CD- MECC is shown to be a simple and attractive approach for (i) the confirmation of the stereoselectivity of the aromatic hydroxylation of mephenytoin and phenytoin, (ii) the simple and rapid differentiation between extensive and poor metabolizers for mephenytoin, and (iii) assessment of compliance.

Buffers↗

PubMed1994

Assessment of impact of physico-chemical drug properties on monitoring drug levels by micellar electrokinetic capillary chromatography with direct serum injection.

The impact of physico-chemical properties of 25 compounds, including antiepileptic, anti-inflammatory and beta-blocking drugs, on their determination by micellar electrokinetic capillary chromatography (MECC) with direct serum injection (DSI) is discussed. Having a pH 9.2 buffer containing 75 mM sodium dodecyl sulfate (SDS), elution is dependent on hydrophobicity, the order of emergence being basically according to increasing octanol/water partition coefficients (logP values). Peak shape is determined by the dissociation behavior (expressed by pKa) and plasma protein binding (PPB). Sharp peaks are produced by compounds having low PPB and, independently of PPB, by drugs with pKa values which are similar to the buffer pH. Broad or double peaks are established by drugs of low pKa values and significant (> about 40%) PPB. In order to evaluate the effective amount of a protein-bound drug measured by MECC-DSI, serum levels of drugs with different PPB, namely ethosuximide (no PPB), phenobarbital (PPB of about 50%) and naproxen (PPB > 99%) have been determined by both MECC-DSI and MECC with extract injection (MECC-EXI). In each case, with more than 40 sera, there is good agreement between the two sets of data. Thus, employing MECC-DSI, total amounts of drugs are determined, i.e. a complete release of the drugs from the proteins is effected by the impact of dodecyl sulfate on the sampled proteins.

Adrenergic beta-Antagonists↗

PubMed1994

Rapid determination of the antimycotic drug flucytosine in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

The rapid determination of flucytosine (5-FC) in human serum by micellar electrokinetic capillary chromatography (MECC) with direct sample injection is discussed. Minute (nanoliter) quantities of patient sera are applied to the beginning of a fused silica capillary filled with a phosphate/borate buffer (pH 9.2) containing 75 mM sodium dodecyl sulfate. Upon application of an electric field along the capillary, endogenous and drug substances are transported toward the cathode and separated into distinct zones that are detected by on-column ultraviolet absorption. Depending on the capillary and instrument used, 5-FC is shown to elute within 2-3.5 min of current application and free of endogenous interferences. 5-FC becomes also well separated from another often coadministered antimycotic drug, amphotericin B. Thus, quantitation of 5-FC is accomplished without any sample pretreatment. MECC data of 60 patient sera produced on two different automated instruments and 5-FC serum levels obtained by an agar-based bioassay are shown to agree well. For MECC, intraday and interday reproducibility data (80 micrograms/ml level) are shown to be < or = 4.5 and < 7%, respectively. Due to short run times and short capillary equilibration time intervals between runs, a sample throughput of 15/h is feasible. Thus, this technology is suitable for rapid determination of 5-FC serum levels, the time requirement for running a complete calibration, two control sera, and several patient samples being approximately 1 h only.

Calibration↗

PubMed1993

Assessment of automated capillary electrophoresis for therapeutic and diagnostic drug monitoring: determination of bupivacaine in drain fluid and antipyrine in plasma.

In an effort to evaluate the use of electrokinetic capillary technology for therapeutic and diagnostic drug monitoring, samples were analysed batchwise with an automated, high-throughput capillary electrophoretic instrument coupled to an inexpensive PC data acquisition and evaluation system. Examples studied included the capillary electrophoretic (HPCE) determination of bupivacaine in drain fluid collected after pulmonary surgery and the micellar electrokinetic capillary chromatographic (MECC) determination of antipyrine in human plasma. Analyses for antipyrine could be accomplished without any sample pretreatment whereas bupivacaine required extraction prior to analysis. Antipyrine determination was effected through external calibration using either peak areas, relative peak areas or peak heights. The intraday and interday reproducibilities (n = 15) of the evaluated concentrations were 1.5-3% and 5-6%, respectively. For bupivacaine, determination based on internal and external calibration employing peak areas and peak heights was investigated. The intraday and interday reproducibilities (n = 5) of bupivacaine concentrations were about 1% and 2%, respectively, for internal calibration and both about 5% for external calibration. The electrokinetic capillary data compared well with data obtained by gas chromatography (bupivacaine) and high-performance liquid chromatography (antipyrine).

Antipyrine↗

PubMed1993

Determination of morphine-3-glucuronide in human urine by capillary zone electrophoresis and micellar electrokinetic capillary chromatography.

Attempts to determine morphine-3-glucuronide (MO3G) by high-performance capillary electrophoresis and micellar electrokinetic capillary chromatography are reported. Using direct injection of urine, it was possible to achieve a limit of detection of about 20 micrograms/ml, which is poor compared with high-performance liquid chromatography and immunoassays. However, employing sample extraction with C8 cartridges, the presence of MO3G in urines that tested positive for opioids using a commercial enzyme-multiplied immunoassay technique could be successfully confirmed. The limit of detection with unambiguous identification of MO3G via spectral analysis was about 1 microgram/ml.

Buffers↗

PubMed1993

Comparative use of three electrokinetic capillary methods for the determination of drugs in body fluids. Prospects for rapid determination of intoxications.

Three electrokinetic capillary methods, micellar electrokinetic capillary chromatography, capillary zone electrophoresis and capillary isotachophoresis, are shown to be well suited for the rapid screening and confirmation of drugs in serum and urine of patients with medical drug overdoses (intoxications), situations where rapid identification without precise quantification is needed. Patients' samples obtained from the emergency care unit were analysed in an instrument featuring on-column, fast forward-scanning multi-wavelength detection and the data were compared with those obtained by conventional methods. The drugs studied included salicylate, acetaminophen (paracetamol) and antiepileptics. In cases with high drug concentrations, body fluids can be injected directly or may have only to be diluted (urine) or ultrafiltered (serum) prior to analysis, providing results within about 30 min. Thus, electrokinetic capillary methods can be employed for rapid drug screening, provided that instrumentation with a database for peak identification is available.

Acetaminophen↗

PubMed1993

Strategies for the monitoring of drugs in body fluids by micellar electrokinetic capillary chromatography.

Electrokinetic capillary techniques can exploit numerous separation principles, making them flexible and easily applicable to a variety of separation problems. In recent publications, this emerging technology has been shown to be well suited for monitoring drugs and metabolites in body fluids, including serum, saliva and urine. Most attention has been focused on micellar electrokinetic capillary chromatography (MECC) because it permits the separation and determination of drugs with discrimination being largely based on differences in hydrophobicity. An overview of literature data on the MECC of drugs in body fluids and recent data obtained with antiepileptics in serum and saliva, with model mixtures of illicit drugs, and with extracts from urine specimens that tested positively for opiates and cocaine metabolites are presented. Emphasis is focused on buffer selection and simple sample preparation procedures, including direct injection of body fluids, ultrafiltration and solid-phase extraction.

Chromatography, Liquid↗