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W Thormann

Publications and source records attributed to W Thormann.

At least 91 records · Page 5Linked to original sources
PubMed1993

Determination of phenobarbital, ethosuximide, and primidone in human serum by micellar electrokinetic capillary chromatography with direct sample injection.

The determination of antiepileptic drugs in human serum by micellar electrokinetic capillary chromatography (MECC) with direct sample injection is discussed. 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 dodecylsulfate. Upon application of an electric field along the capillary, endogenous and drug substances are transported toward the cathode and separate into distinct zones which are detected by on-column UV absorption. Phenobarbital, ethosuximide, and primidone are shown to elute in front of the solubilized proteins, thus permitting quantitation of these drugs without any sample pretreatment. For phenobarbital and ethosuximide, MECC data obtained using the external standard method and peak areas as the basis for quantitation are shown to be in excellent agreement with those of nonisotopic immunoassays and, for ethosuximide, also with those of high-performance liquid chromatography. The correlation coefficients (n = 50) are between 0.972 and 0.986. Intraday and interday reproducibility data are 2.0-4.5% and 4.5-8.0%, respectively. For primidone, insufficient samples have been available for a comprehensive comparison of MECC data with those of other analytic techniques.

Anticonvulsants↗
PubMed1992

Drug of abuse confirmation in human urine using stepwise solid-phase extraction and micellar electrokinetic capillary chromatography.

This paper demonstrates that most common drugs of abuse can be absorbed simultaneously onto a mixed-mode bonded-phase matrix and eluted sequentially in two to three steps for subsequent analysis by micellar electrokinetic capillary chromatography (MECC). Having on-column multiwavelength UV absorption detection, this is shown to be an attractive approach for confirmation testing of barbiturates, hypnotics, amphetamines, opioids, benzodiazepines, and metabolites of cocaine in a single aliquot of human urine. For these compounds, no hydroysis of the urine specimen or sample derivatization is required. Under the examined conditions using 5 mL of urine, excellent recoveries (80-90% level) and detection limits (about 100 ng/mL) are obtained. For patient urines which tested positively for different classes of drugs using immunological screening methods, a two-step extraction scheme is shown to provide extracts suitable for rapid MECC confirmation of the drugs of abuse.

Chromatography↗
PubMed1992

Confirmation testing of 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid in urine with micellar electrokinetic capillary chromatography.

The major urinary metabolite of the most commonly abused psychotropic drug, delta-9-tetrahydrocannabinol, is 11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid (THC-COOH). With basic hydrolysis, extraction and concentration, this compound can easily be determined using micellar electrokinetic capillary chromatography with on-column multi-wavelength detection. After solid-phase extraction of 5 ml of urine, drug concentrations down to about 10 ng/ml can be unambiguously monitored. Peak assignment is achieved through comparison of the retention time and absorption spectrum of the eluting THC-COOH peak with those of computer-stored model runs. The effectiveness of the approach is demonstrated with data obtained from urine samples from different patients which tested positively for cannabinoids using a fluorescence polarization immunoassay.

Cannabinoids↗
PubMed1992

Determination of substituted purines in body fluids by micellar electrokinetic capillary chromatography with direct sample injection.

Many substituted purines (theobromine, caffeine, paraxanthine, theophylline and uric acid, as well as other methylated xanthines and uric acids) can easily be separated and analysed in one run using micellar electrokinetic capillary chromatography with a boratephosphate buffer containing 75 mM sodium dodecyl sulphate (pH approximately 9). Serum, saliva and urine samples collected after the self-administration of caffeine and serum samples from patients receiving theophylline or caffeine pharmacotherapy were screened for substituted purines. The data presented show the ease of using on-column multi-wavelength detection for investigating the feasibility of direct sample application, the characterization of sample pretreatment procedures and peak confirmation by comparing absorption spectra. It is shown that the determination of purines in serum and saliva samples, including therapeutic concentrations of caffeine and theophylline, can be accomplished without any sample pretreatment, whereas sample extraction is required for the determination of purines in urine. Quantitative data for the determination of micromolar amounts of theophylline (samples from adult patients) and caffeine (samples from infants born prematurely) in serum samples compared well with data obtained by non-isotopic immunoassays. Micellar electrokinetic capillary chromatography with the direct injection of serum or saliva samples requires only microlitre volumes of sample and several different compounds can be determined within a few minutes.

Adult↗
PubMed1992

Evaluation of instrumental, nonisotopic immunoassays (fluorescence polarization immunoassay and enzyme-multiplied immunoassay technique) for cyclosporine monitoring in whole blood after kidney and liver transplantation.

Two new instrumental methods, an enzyme-multiplied immunoassay technique (EMIT) and a fluorescence polarization immunoassay (FPIA), were evaluated for monitoring of cyclosporine (CyA) in whole blood samples of renal and liver transplant patients. They are considered as being specific to the parent drug and they were compared with a specific radioimmunoassay (RIA) and a nonspecific FPIA. The data reveal that the novel procedures provide slightly overestimated CyA levels compared with specific RIA (6-12% for EMIT, 20-25% for FPIA). For both assays, intrarun and interrun reproducibilities were found to be in the 2-8% range. The ease of performance and the possibility of performing approximately 40 assays/h make the two methodologies very attractive for both routine and emergency analyses. These approaches are viewed to be complementary to the only previously available instrumental method, the nonspecific FPIA, which provides three- to fourfold higher CyA levels than those obtained with specific methods. Specific and nonspecific monitoring of CyA levels allowed variations in proportions of metabolites to total CyA and metabolites to be distinguished. A higher percentage and variability of cross-reacting metabolites were found in whole blood samples after liver transplantation compared with those in blood of kidney transplant recipients.

Cyclosporine↗
PubMed1991

Analysis of illicit drugs in human urine by micellar electrokinetic capillary chromatography with on-column fast scanning polychrome absorption detection.

Using micellar electrokinetic capillary chromatography (MECC) with a borate/phosphate buffer containing 75 mM SDS (pH 9.1), common drugs of abuse and/or their metabolites, including opioids, benzoylecgonine, amphetamines, and methaqualone, can easily be analyzed. After solid-phase extraction of 5 mL of urine, drug concentrations down to about 100 ng/mL can be unambiguously monitored with on-column multiwavelength detection. Peak assignment is achieved through comparison of retention times and absorption spectra of eluting peaks with those of computer-stored model runs. The effectiveness of the approach is demonstrated with data obtained from different patient urines which tested positively for one or several drugs using nonisotopic immunoassays. Results suggest that MECC of illicit drugs is a highly specific and sensitive instrumental approach suitable for confirmation testing following a positive response of a toxicological screening procedure.

Absorption↗
PubMed1991

Purification of ovalbumin and lysozyme from a commercial product by recycling isotachophoresis.

The aim of this work was to test the suitability of using recycling isotachophoresis (RITP) for the purification of ovalbumin (OVA) and/or lysozyme (LYSO) from a commercial OVA product containing LYSO and conalbumin (CAL) as major proteinaceous impurities. The search for suitable electrolyte systems and spacers was carried out by capillary isotachophoresis. RITP was performed in a recycling free-flow focusing apparatus in the batch mode with immobilization of the advancing zone structure via a controlled counterflow. Typically 700 mg of the commercial product were processed within 2 h. Enhancement of the sample load was achieved by a feed of sample under counterflow control. The collected fractions were analysed separately for conductivity, pH and ultraviolet absorption, and selected fractions were characterized by analytical capillary electrophoretic methods. All three proteins could be separated and fractionated using suitable spacers. Depending on the chosen conditions either OVA or LYSO could be purified in amounts larger than milligrams per hour (OVA 300 mg/h; LYSO 10 mg/h). The instability of CAL in solution prevented its isolation in the investigated configurations.

Conalbumin↗
PubMed1991

Determination of thiopental in human serum and plasma by high-performance capillary electrophoresis-micellar electrokinetic chromatography.

The quantitation of thiopental in human serum and plasma was investigated using high-performance capillary electrophoresis (HPCE) in a micellar configuration and the results were compared with reversed-phase high-performance liquid chromatography (HPLC). Thiopental and an internal standard (carbamazepine for HPCE and thiamylal for HPLC) were extracted from serum or plasma using pentane and a phosphate buffer (pH 6.4). HPCE analysis took place in a phosphate-borate buffer with 50 mM sodium dodecyl sulphate using an automated instrument and HPLC was performed with a C8 column and a mobile phase of phosphate buffer-acetonitrile (65:35, v/v). HPCE and HPLC data from 66 patient samples compared well based on linear regression analysis. However, estimates obtained with the inclusion of the internal standard were lower than those based on the sample peak only. This example allows the elucidation of the advantages of using HPCE as an assay methodology for the therapeutic monitoring of thiopental and other drugs.

Chromatography↗
PubMed1991

Isotachophoresis of proteins in uncoated open-tubular fused-silica capillaries with a simple approach for column conditioning.

The isotachophoretic determination of proteins in uncoated open-tubular fused-silica capillaries of 50 and 75 microns I.C. with on-column multi-wavelength detection is reported. Small amounts of hydroxypropylmethylcellulose added to the leader provide an efficient method of dynamic column conditioning which allows the high-resolution isotachophoretic determination of most proteins to be performed in the presence of an electro-osmotic flow. Different approaches for cationic and anionic analyses are discussed and illustrated with selected examples.

Animals↗
PubMed1991

Recycling and screen-segmented column isotachophoresis, two free-fluid approaches for fractionation of proteins.

Recycling and screen-segmented column isotachophoresis (ITP), two approaches for the milligrams to grams preparative-scale purification of proteins, are discussed and compared. Recycling ITP was performed in a recycling free-flow focusing apparatus. In this process, fluid flows rapidly through a narrow channel and the effluent from each channel is reinjected into the electrophoresis chamber through the corresponding input port. The residence time in the cell is of the order of 1 s per single pass, which does not allow complete separation, so recycling is essential to attain the steady state. Immobilization of the advancing zone structure is obtained via a controlled counterflow. Thirty fractions of about 4 ml each are obtained. Column ITP was executed in a Rotofor apparatus and in a similar column operated vertically and without rotation. These instruments feature a screen-segmented annular separation space with twenty subcompartments of about 2 ml each. With both approaches, the collected fractions were analysed separately for conductivity, pH and UV absorbance. Selected fractions were characterized by analytical electrophoretic methods. Examples presented include the cationic and anionic ITP behaviour of model proteins, including bovine serum albumin, ovalbumin and ribonuclease A, and the ITP removal of the major impurities from a commercial ovalbumin sample. These examples revealed that the screen-segmented column is suitable for ITP protein purification and operates optimally in a horizontal rotating mode and without internal cooling. The recycling experiments showed that counterflow improves separation and the steady-state patterns are dependent on the fluid layer thickness in the separation cell but, with a given gap, essentially independent of applied current and recycling pump rate.

Electrophoresis↗
PubMed1991

Analysis of barbiturates in human serum and urine by high-performance capillary electrophoresis-micellar electrokinetic capillary chromatography with on-column multi-wavelength detection.

The analysis of barbiturates in human serum (or plasma) and urine by high-performance capillary electrophoresis-electrokinetic capillary chromatography with on-column fast-scanning multi-wavelength detection is discussed. The use of a buffer of ca. pH 8 and containing sodium dodecyl sulphate provides a medium suitable for fast and high-resolution separations of barbiturates. Seven barbiturates are characterized by their retention and absorption spectra between 195 and 320 nm. Comparison of these computer-stored data with those of unknown samples is shown to allow the identification of barbiturates in samples of patients undergoing pharmacotherapy and in toxicological urine and serum specimens. Three-dimensional electropherograms provide reliable information on the requirement and suitability of sample pretreatment procedures. With urine, extraction of barbiturates prior to analysis is necessary. With human serum several barbiturates, including phenobarbital, are shown to elute in an interference-free window in front of uric acid and the proteins, allowing these substances to be determined by direct sample injection. The need for multi-wavelength detection over a relatively wide wavelength range as a means of peak confirmation in electrokinetic capillary analyses is demonstrated and limitations of this technique for compounds with similar retention behaviour and absorption spectra are discussed.

Barbiturates↗
PubMed1991

Innovative developments in isotachophoresis (displacement electrophoresis).

This Mini Review is aimed at characterizing the innovative developments in isotachophoresis (ITP) during the past few years, discussing in turn new theoretical, analytical, preparative and applicative aspects of this unique separation method. Examples given from our laboratory include the study of the detailed dynamics of the ITP separation of four components by computer simulation and experimental validation in a capillary-type instrument with multiple sensors along the separation trough; the anionic ITP analysis in presence of a strong cathodic electroosmotic flow using an open-tubular fused-silica capillary with on-column multiwavelength detection, and the fractionation of proteins in a screen-segmented, rotating column as well as by recycling ITP.

Electrophoresis↗
PubMed1990

Experimental and theoretical description of the isotachophoretic behavior of serum albumin.

The isotachophoretic behavior of serum albumin is examined for three anionic and one cationic electrolyte systems by (i) computer simulation, (ii) capillary isotachophoresis (ITP) and (iii) continuous flow ITP. The theoretical relationship between pH of the leading electrolyte and the steady state protein plateau concentration is presented for one of the anionic systems. With leading ion concentrations of the order of 10 mM, experimental protein plateau concentrations of 1.3-2.3% w/v are obtained. The computer predictions are approximately half these values.

Aminocaproic Acid↗
PubMed1990

Isotachophoretic zone formation of serum albumin in different free fluid electrophoresis instruments.

The isotachophoretic behavior of a model protein, serum albumin, was examined (i) by computer simulation, (ii) by capillary isotachophoresis in HPE 100 and Tachophor 2127, (iii) by continuous flow isotachophoresis in Elphor VaP 22 and the BIO-STREAM Separator and (iv) by recycling isotachophoresis in an apparatus of our own design. Variations in monitored zone shapes can be explained by differences in engineering aspects and fluid stabilization principles of the instruments.

Cations↗
PubMed1990

Experimental and theoretical dynamics of isoelectric focusing: IV. Cathodic, anodic and symmetrical drifts of the pH gradient.

The production of anodic, cathodic and symmetrical drifts of a pH 3.5-10 gradient formed by isoelectric focusing in polyacrylamide gels is demonstrated experimentally by manipulation of the electrolyte concentrations. Experimental behavior is reproduced by computer simulation of a model mixture of 15 hypothetical carrier ampholytes whose pIs span the pH range 3-10. The mechanism which produces the drifts is elucidated and approaches to minimize such drifts are discussed. The data suggest why most experimentally observed drifts are cathodic.

Computer Simulation↗
PubMed1990

Capillary electrophoretic determination of S-carboxymethyl-L-cysteine and its major metabolites in human urine: feasibility investigation using on-column detection of non-derivatized solutes in capillaries with minimal electroosmosis.

The capillary electrophoretic analysis of S-carboxymethyl-L-cysteine and some of its metabolites in human urine is reported using (i) on-column detection of underivatized solutes, (ii) minimal sample pretreatment and (iii) capillary columns with minimized electroosmosis. Experimental results obtained with two apparatus, the HPE-100 (Bio-Rad), featuring coated fused silica capillaries of 25 microns i.d., and with the Tachophor 2127 (LKB), having Teflon capillaries of 500 microns i.d. are discussed. Drug concentrations down to 0.2 mg/mL on capillary isotachophoresis and 0.03 mg/mL on capillary zone electrophoresis can be monitored with these instruments.

Carbocysteine↗
PubMed1989

Computer simulation and experimental validation of the electrophoretic behavior of proteins.

A mathematical model of the electrophoretic behavior of proteins is presented. The Debye-Hückel-Henry theory is used for the description of protein mobility, which has the important result of making net mobility a function of ionic strength. A net charge vs pH relationship and a diffusion coefficient are required to describe a specific protein. The model is employed for the computer simulation of three distinct electrophoretic modes: isoelectric focusing, isotachophoresis, and zone electrophoresis. The validity of the model is tested by comparing simulation with experimental data. Excellent qualitative agreement was found.

Computer Simulation↗