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Separation of cresols using coelectroosmotic capillary electrophoresis.

A new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire-resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolution.

1-Propanol↗
PubMed1993

Analysis of leukotrienes, prostaglandins, thromboxane B2 and their metabolites by capillary isotachophoresis.

An analytical capillary isotachophoretic method for the analysis of the eicosanoids leukotriene E4, leukotriene B4, prostaglandins E1 and E2, 6-keto-prostaglandin F1 alpha, thromboxane B2, and their metabolites of omega- and/or beta-oxidation is described. The method is based on anionic separation and detection by UV absorbance (254 nm) and conductivity and allows simultaneous analysis of the primary compounds and their corresponding major urinary metabolites. The method was applicable to the qualitative and quantitative analysis of prostaglandin E1 in a drug preparation.

6-Ketoprostaglandin F1 alpha↗
PubMed1993

Analysis of plasmid-DNA and cell protein of recombinant Escherichia coli using capillary gel electrophoresis.

Plasmid DNA prepared from cultivation samples of recombinant Escherichia coli was analyzed by capillary gel electrophoresis. We used this method to control the genetic stability during a fed-batch culture. The plasmid DNA and a standard DNA mixture with molecular weights in the size range of 3000-22,000 base pairs (bp) were analyzed in capillaries that were filled with solutions of non-cross-linked polyacrylamide. With this method the plasmid DNA from recombinant E. coli cultivation samples was analyzed within 30 min. Separation parameters such as gel concentration, capillary length and current were optimized for that purpose. The method was modified to allow the analysis of proteins. Crude cell lysate samples could be screened for the protein pattern within 15 min in sodium dodecyl sulfate-polyacrylamide filled capillaries. The method was also used to verify product purity after the down-stream process.

Capillary Action↗
PubMed1993

Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix.

Obtaining accurate molecular weight estimates for glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been difficult due to the lack of SDS binding by the carbohydrate moieties of the proteins. This leads to lower charge-to-mass ratios for SDS-glycoprotein complexes, resulting in over-estimation of molecular weights by SDS-PAGE. In order to minimize these inaccuracies for proteins with abnormal charge-to-mass ratios, a Ferguson plot may be employed. This application requires the determination of relative mobilities for standard proteins in addition to unknowns at several different gel concentrations. Historically, this technique has not been popular because it requires time-consuming preparation of gels with varying matrix concentrations, electrophoresis, and staining/destaining of gels. In this paper a procedure is demonstrated which automatically generates all of the data required for a Ferguson plot using a replaceable sieving matrix (thereby eliminating gel polymerization) in a capillary format. In addition, this technique possesses the advantages inherent to capillary electrophoresis, namely, very fast separation times, and on-line monitoring which allows quantitation and precludes post-separation staining and destaining of gels.

Avidin↗
PubMed1993

The variance and the quantity 2Dt in capillary zone electrophoresis.

The variance is generally used as a measure of dispersion of zones in capillary zone electrophoresis (CZE). It is a quantitative measure of the separation power of a system and different causes of dispersion can be rated by their partial variances, which can be summed up to total variance. However, the additivity is only valid for independent dispersion sources, a fact that often seems to be ignored. The ubiquitous dispersion source diffusion is taken into consideration by the Einstein term 2Dt. Other sources of dispersion are, e.g., injection, detection, thermal gradients, adsorption, and hydrodynamic flow. For each of these sources various variance expressions have been derived. The origin of the term 2Dt and its relation to the variance is explained and the calculation of variance in general is discussed. The equivalence of the diffusion variance and the term 2Dt is verified with some simple initial forms of sample zone and the additivity of variances in ideal zone electrophoresis is demonstrated. The change of conductivity in zones results in asymmetrical zone forms which is an indication of nonideality of a system. It is shown that in such cases the term 2Dt is no longer valid and its use as an additive variance component leads to an erroneous total variance. Because in zone electrophoresis conductivity in a zone always changes more or less, the additivity of variances is never perfectly valid. However, in many cases the nonideality may be so small that the additivity in practice is still applicable.(ABSTRACT TRUNCATED AT 250 WORDS)

Capillary Action↗
PubMed1993

On the kinetics of monomer incorporation into polyacrylamide gels, as investigated by capillary zone electrophoresis.

The kinetics of monomer incorporation into a polyacrylamide gel have been studied in a photopolymerization system comprising 100 microM methylene blue in presence of a red-ox system, 1 mM sodium toluenesulfinate (reducer) and 50 microM diphenyliodonium chloride (oxidizer). A precise assessment of gel point (pc) was obtained in a droplet chamber, in which argon was gently bubbled with a fused silica capillary into the reaction mixture. At pc, 50% (+/- 3) acrylamide was incorporated into the matrix, vs. 80% (+/- 4) N,N'-methylenebisacrylamide. This incorporation level remained the same when polymerized in the 2-36 degrees C temperature range. Incorporation continued almost linearly for acrylamide up to 80% conversion. The reaction was continued up to 55 min (at 2 degrees C), at which point bisacrylamide had been essentially consumed (> 99.5% incorporation) and acrylamide had reacted (95%). At 2 degrees C, after gelation, the gel became progressively turbid (the Tyndall effect plateauing at 50 min), but it remained fully transparent if, at the gel point, reaction was continued at 50 degrees C. The consumption of the pendant double bonds of Bis followed the progression of turbidity. It is concluded that, by gelation at 2 degrees C, the nascent chains form clusters held together by hydrogen bonds (melting point at 28 degrees C); such clusters are subsequently "frozen" in the three-dimensional space as the pendant double bonds in the chains react progressively. Such turbid matrices are more porous and less elastic than when the gel is polymerized at 50 degrees C. This process is similar to the "lateral aggregation" occurring when gels are formed in presence of a polymer in solution (e.g. 10 KDa polyethylene glycol; Righetti et al., Electrophoresis 1992, 13, 587-594).

Acrylic Resins↗
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

Characterization of proteins by capillary electrophoresis in fused-silica columns: review on serum protein analysis and application to immunoassays.

Protein mixtures can be characterized in terms of their separations by capillary electrophoresis (CE). The separation of proteins by CE is performed in untreated fused-silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separated in such columns in less than 10 min in the presence of phosphate buffer with a pH between 4.0 and 9.0. The application of CE separation procedures for routine analysis of protein in serum, urine, and cerebrospinal fluid in borate-based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10(-5) M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one component is labeled. Thus, an antigen analyte, or the antibody to the analyte, may be labeled with a fluor and detected by laser-induced fluorescence (LIF). With a fluorescent-labeled reactant, the use of LIF detection further extends the detection limit to 10(-11) M. The CE separation technique for proteins provides a means to separate the bound and free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conjunction with laser-induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations in the range of 10(-9) to 10(-10) M is shown.

Blood Proteins↗
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

Analysis of lipoproteins with analytical capillary isotachophoresis.

An analytical free flow capillary isotachophoresis procedure, with a discontinuous electrolyte system, for the detailed analysis of lipoproteins in human body fluids has been developed. The technique is based on prestaining whole serum lipoproteins with a lipophilic dye before separation. Human serum lipoproteins are separated into 14 well-characterized subfractions according to their electrophoretic mobility. High density lipoproteins (fraction 1 to 6) are separated into three major subpopulations, the fast migrating high density lipoprotein (HDL) subpopulation, containing mainly apo AI and phosphatidylcholine, the subpopulation with intermediate mobility, consisting of particles rich in apo AII, apo E, and C apolipoproteins, and the slowly migrating HDL subfraction, containing mainly particles rich in apo AI, apo AIV, and lecithin: cholesterol acyltransferase (LCAT) activity. The apo B containing lipoproteins (fraction 7 to 14) can be subdivided into four major functional groups. The first represents chylomicron derived particles and large triglyceride-rich very low density lipoproteins (VLDL). The second group consists of small VLDL and intermediate density lipoprotein (IDL) particles, anf the third and fourth group represent the low density lipoproteins. The isotachophoretic analysis of human serum samples obtained from patients with hyperlipoproteinemias is compatible with the classification according to the Frederickson phenotypes and reflects the respective biochemical abnormalities. Furthermore, several genetic disorders of lipid and lipoprotein metabolism like HDL deficiency syndromes, familial LCAT deficiency, Fish eye disease, hypobetalipoproteinemia and abetalipoproteinemia can be well characterized by analytical capillary isotachophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoproteins↗
PubMed1994

Determination of 2-5A synthetase and 2-5A phosphodiesterase in neuroblastoma cells by analytical capillary isotachophoresis: effects of cytokines and comparison with radioenzymatic methods.

2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.

2',5'-Oligoadenylate Synthetase↗
PubMed1994

Detection of neurotensin in tissues by capillary zone electrophoresis.

An electrophoretic method for detecting neurotensin in tissues was developed. Homogenates of rat duodenum and adrenal glands were first extracted by solid-phase extraction and purified by reversed-phase high-performance liquid chromatography. The neurotensin-rich fraction was further analyzed by capillary zone electrophoresis (CZE) using a commercial instrument with UV detection. A minor peak was detected as neurotensin and its identity was further confirmed by performing several CZE analyses at different pH values. Such an approach could be used to analyze with good molecular specificity the neuropeptides in some human tissues.

Adrenal Glands↗
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

Investigation of drug metabolism using capillary electrophoresis with photodiode array detection and online mass spectrometry equipped with an array detector.

The application of capillary electrophoresis (CE) with photodiode array detection (DAD) and on-line CE-mass spectrometry (CE-MS) equipped with a position and time resolved (PATRIC) focal plane detector for analysis of both in vitro and in vivo drug metabolism is demonstrated. Separation of metabolites derived from the neuroleptic drug haloperidol, by CE, using a simple, volatile run buffer containing 50 mM ammonium acetate with 10% methanol and 1% acetic acid is reported. The potential utility of CE-DAD for screening drug metabolite mixtures derived from hepatic microsomal incubations is demonstrated for haloperidol (HAL). Also the potential problems associated with using this technology to screen human urine samples for HAL metabolites is discussed. Furthermore, the usefulness of CE-MS and CE-electrospray ionization skimmer collision induced dissociation-MS (CE-ESI-CID-MS) in identification and structure elucidation of HAL metabolites derived from both a guinea pig hepatic microsomal incubation and urine from a patient treated with 0.5 mg/day of HAL is shown. The utility of such an approach in the general area of clinical pharmacology is also discussed.

Animals↗
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

Pharmaceutical and biomedical applications of capillary electrophoresis/electrochemistry.

The use of capillary electrophoresis/electrochemistry (CEEC) for the analysis of microdialysis samples obtained for pharmacokinetic and neurochemical studies is described, as well as the development of new types of electrodes and waveforms which increase the selectivity of this technique for specific classes of analytes. CEEC with a carbon fiber electrode was employed for the analysis of microdialysis samples. Microdialysis is an in vivo sampling technique that yields very small samples for analysis (less than 1 microL). Therefore, capillary electrophoresis, with its small volume requirements, is an excellent choice for the analytical method. CEEC was used to study the pharmacokinetics of L-dopa and the release of aspartate and glutamate following a high K+ infusion in the brain. Several modified electrodes which increase the applicability of CEEC in pharmaceutical and biomedical analysis are described. One of these is a gold/mercury electrode which is highly selective for thiols and was used for the determination of glutathione in a rat brain. An alternative method for the detection of thiols employed a chemically modified electrode containing cobalt phthalocyanine. In this case, an electrocatalyst reduces the overpotential of thiols at the carbon electrode and makes it possible to detect them at a much lower and more selective oxidation potential. This electrode was used for the detection of cysteine in urine. The development of pulsed amperometric detection for capillary electrophoresis is also described and is demonstrated by the detection of glucose in blood. Lastly, a method for the detection of peptides based on the formation of a copper complex and detection at a carbon fiber electrode is discussed.

Animals↗
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↗