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PubMed1998

Capillary electrochromatography of derivatized mono- and oligosaccharides.

An octadecyl-silica (ODS) stationary phase with light surface coverage of octadecyl ligands was introduced for capillary electrochromatography (CEC) at moderate electroosmotic flow (EOF) velocity. The ODS stationary phase was intentionally produced with light surface coverage in order to ensure a moderate EOF velocity across the packed capillary column, thus allowing relatively rapid analysis time. Despite the fact that the stationary phase leaves 75% of the surface silanols unreacted, fused-silica capillary columns packed with this ODS stationary phase exhibited reversed-phase behavior toward neutral alkylbenzene homologous solutes using hydroorganic eluents. Closely related p-nitrophenylglycosides including some p-nitrophenyl-monosaccharides and p-nitrophenyl-maltooligosaccharides were readily separated on the ODS capillary column within a relatively short analysis time. Also, alpha- and beta-anomers of some p-nitrophenyl-monosaccharides were readily separated in the presence of a small amount of borate buffer in the hydroorganic eluent.

Acetonitriles↗
PubMed1998

Capillary electrochromatography with novel stationary phases. I. Preparation and characterization of octadecylsulfonated silica.

A novel silica-based stationary phase was developed for use in capillary electrochromatography (CEC) at relatively high electroosmotic flow (EOF). The silica was first bonded with a relatively hydrophilic layer bearing strong sulfonic acid groups. To this charged polar sublayer, octadecyl functions were covalently attached to yield the nonpolar top layer. This novel stationary phase, referred to as octadecylsulfonated silica (ODSS), was packed in bare fused-silica capillaries or in capillaries with the same coating as the sublayer on the silica-based stationary phase. The resulting packed columns were evaluated in CEC using alkylbenzenes as the test model solutes. Good separations can be achieved in less than 8 min, much faster than when using a regular octadecyl silica capillary column. Due to the permanent negative charge provided by the sulfonated sublayer on both the capillary walls and the silica particles, the magnitude of the EOF remained more or less constant over a wide range of pH, and its magnitude can be conveniently varied by the applied voltage.

Benzene↗
PubMed1998

Method development in pharmaceutical analysis employing capillary electrochromatography.

Capillary electrochromatography (CEC) has been employed to explore method development for a series of structurally related polar neutral compounds of pharmaceutical relevance. Capillaries with dimensions of 75 microm ID x 25 cm length (34.5 cm total) were packed with Spherisorb ODS-1, Hypersil phenyl, and Hypersil MOS (all 3 microm particles) and were compared in the reversed-phase mode in order to determine which phase provided the best initial performance and thus serve as the phase of choice for additional method development experiments. The various separation parameters examined for their effect on efficiency, k', resolution, and linear velocity included percent and type of organic modifier, buffer concentration, voltage, and temperature. All separations were conducted with an acidic mobile phase (aqueous mobile phase component, pH 3.0). The separation efficiencies obtained were on the order of 200,000-260,000 plates/m, which equates to reduced plate heights of 1.22 for columns packed with Spherisorb ODS-1. Repeatable column-to-column separation performance was demonstrated.

Acetonitriles↗
PubMed1998

Separation of neutral compounds by capillary electrokinetic chromatography using polyethyleneimine as replaceable cationic pseudostationary phase.

Polyethyleneimine (PEI, molecular weight 6 x 10(5) - 1 x 10(6)) is applied as a positively charged pseudostationary phase for electrokinetic chromatography (EKC) of uncharged mono- and oligophenols. EKC is carried out in PEI-coated fused-silica capillaries (with electroosmotic flow directed towards the anode) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.0, 20 mM) with PEI added to the solution in concentrations up to 0.70% w/v. The pseudostationary phase leads to a retardation of the solutes mainly according to the number (and the position) of the OH-groups of the separands, and is not influenced significantly by methyl groups. For 0.70% w/v PEI solution, for instance, the relative retention, rho, has values between 0.33 and 0.53. For the systems with the highest resolution of the separands (0.25-0.30% PEI) 190,000 plates per meter are observed. The results indicate that the separation selectivity is mainly caused by ion-dipole interactions between the OH-groups of the solutes and the pseudostationary phase.

Capillary Action↗
PubMed1985

In situ replication techniques: II. Quantitative methodologies for replicate materials.

Because replicate materials have requirements different from those of recording or impression materials, quantitative methodologies were sought using commercial impression materials. Two satisfactory objective techniques resulted, a laser-scattering and a capillary flow test. Using high-resolution gratings to stimulate tooth detail (less than 1 micron), the reproduction quality of 36 two-stage replicas was determined in diffraction, reflection, and in an unblazed state. Using precision bore glass tubes (0.25, 0.5, 1, and 2 mm diameters) to simulate the high-energy surface of enamel, the flow characteristics of nine elastomers (the first stage replicates) and four epoxies (the second stage replicates) were determined at isobaric conditions. Because the laser spot size was relatively large (0.6 mm) and the pressure differential was small (25 mm Hg), both the global resolution and the low shear rate characteristics could be measured. Of the commercial materials tested, Reprosil Light had the best combination of fluidity and resolution, regardless of which positive material was used. Although Permagum Low, Silene Wash, and Xantopren Blue scored high in one of the two tests, none of these materials could compare to Reprosil Light within the context described herein.

Capillary Action↗
PubMed1975

Water states and water gates in osmotic processes, and the inoperative concept of molfraction of water.

An historical account is given of concepts regarding the mechanism of osmosis and imbibition, starting with Lord Kelvin's gravitational column, where he pointed out that a capillary standing in a dish of water within an isothermal enclosure must have a lowered vapor pressure at its elevated meniscus so as to match that emanating from the surface in the dish, otherwise distillation would violate the Second law. A brilliant sequence to this simple idea followed through Poynting, Arrhenius, Noyes and culminated with Hulett, who in 1901 formulated the "solvent tension theory" of osmosis, stating in essence that the thermal motion of the solute molecules by impact with the free solvent surface put the solvent under tension. This lowers the vapor pressure and thereby also its freezing point. Perrin, in famous experiments on Brownian motion, demonstrated solute-solvent independence within a solution and further support came through Herzfeld, Mysels and Duclaux. We measured negative pressures in salt-free sap of mangroves and other plants matching the osmotic pressure in the leaf cells. A series of measurements on magnetic and gravitational effects on osmotic pressure likewise bore out the tension theory. The fashionable "water concentration theory" is left experimentally contradicted and in violation of the Second law.

Capillary Action↗
PubMed2006

New designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I): studies on its metabolism and toxicological detection in rat urine using gas chromatographic/mass spectrometric and capillary electrophoretic/mass spectrometric techniques.

Studies are described on the metabolism and the toxicological analysis of the phenethylamine-derived designer drug 4-iodo-2,5-dimethoxy-beta-phenethylamine (2C-I) in rat urine using gas chromatographic/mass spectrometric (GC/MS) techniques, and for a particular question, using capillary electrophoretic/mass spectrometric (CE/MS) techniques. The identified metabolites indicated that 2C-I was metabolized on the one hand by O-demethylation in position 2 and 5, respectively, followed either by N-acetylation or by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. The latter metabolite was hydroxylated in beta-position and further oxidized to the corresponding oxo metabolite. On the other hand, 2C-I was metabolized by deamination with subsequent oxidation to the corresponding acid or reduction to the corresponding alcohol, respectively. 2C-I and most of its metabolites were partially excreted in conjugated form. The authors' systematic toxicological analysis (STA) procedure using full-scan GC/MS after acid hydrolysis, liquid-liquid extraction and microwave-assisted acetylation allowed the detection of an intake of a dose of 2C-I in rat urine that corresponds to a common drug users' dose. Assuming similar metabolism, the described STA procedure should be suitable for proof of an intake of 2C-I in human urine.

Animals↗
PubMed2006

Investigation of low-abundant in vitro metabolites of stable isotope-labelled BAL4815 by accurate mass capillary-LC-ESI-qTof-MS and MS/MS.

The metabolic profile of BAL4815, an antifungal azole drug, was determined using in vitro rat hepatocyte incubations and subsequent analysis by capillary LC-qTof-MS and MS/MS including accurate mass determination. For the detection of the metabolites, a mixture of the drug and its deuterium-labelled analogue was used for incubations. Metabolic stability of BAL4815 was high in cultured rat hepatocytes. However, several low-abundant metabolites were detected by the use of capillary LC-qTof-MS and manual investigation of the data. The peak intensity of the most abundant metabolite was close to the limit of detection. Except for an apparent oxidation product, the masses of the other detected metabolites could not be assigned to a single and frequently occurring biotransformation. Accurate mass determination and possible elemental compositions suggested that metabolism occurred through a combination of glutathionylation and defluorination. This was verified using accurate mass MS/MS. The use of accurate mass measurements and the derived suggestions for the elemental compositions were essential to elucidate this atypical metabolic pathway. A mass accuracy better than 8 ppm could be achieved for most assigned MS and MS/MS signals with intensities less than 6 cps in the spectra.

Animals↗
PubMed2005

Determination of bradykinin and arg-bradykinin in rat muscle tissue by microdialysis and capillary column-switching liquid chromatography with mass spectrometric detection.

Quantification of bradykinin peptides in limited amounts of rat muscle tissue dialysate has been performed using a packed capillary LC-ESI-TOF-MS method. The micro dialysate samples (450 microL) with added internal standard were loaded onto a 1 mm x 5 mm loading column packed with 5 microm Kromasil C18 particles by a carrier solution of 0.1% formic acid in ACN/water (5:95, v/v) at a flow rate of 250 microL/min for online preconcentration of the analytes. Back-flushed elution onto a 150 mm x 0.5 mm Zorbax C18 column packed with 5 microm particles was conducted using a linear solvent ACN/H2O gradient containing 0.1% formic acid. (Tyr8)-bradykinin was used as an internal standard and was added to the dialysis sample prior to injection. Baseline separation of bradykinin, arg-bradykinin and (tyr8)-bradykinin was achieved within 10 min. Positive ESI was performed in the m/z range of 200-1300. The method was validated in the range 0.2-1.0 ng/mL dialysate, yielding correlation coefficients of 0.995 and 0.990 for bradykinin and arg-bradykinin, respectively. The within-assay and between-assay precisions were between 4.3-9.6% and 6.2-10.6%, respectively. Both arg-bradykinin and bradykinin were detected in dialysate from rat muscle tissue, at concentrations of 0.1 and 0.4 ng/mL for bradykinin and arg-bradykinin, respectively, confirming the presence of arg-bradykinin in rat muscles.

Animals↗
PubMed2005

Capillary liquid chromatography and tandem mass spectrometry for the quantification of enkephalins in cerebrospinal fluid.

A capillary LC-MS/MS system was evaluated for the absolute quantification of enkephalins in cerebrospinal fluid (CSF). On column focusing on a C18 trapping column, in-line with the analytical column, was used for preconcentration. Quantification was performed with a triple quadrupole instrument in the multiple reaction monitoring mode. Weighted linear regression analysis proves to be a good linearity in a dynamic range of two orders of magnitude. The method was validated, yielding calibration curves with correlation coefficients greater than 0.9914. Assay precision and accuracy were evaluated by direct injection of enkephalin fortified artificial CSF (aCSF) samples at three concentration levels. Mean accuracy of analysed concentrations was between 97.63 and 107.6%. LOD and LOQ were assessed at, respectively, 0.5 and 1 pmol/mL. Validation results show that it is feasible, with a capillary LC-MS/MS system, to quantify neuropeptides in the low femtomole range in aCSF. The obtained coefficients of variation, however, indicate that the use of appropriate isotopically labelled internal standards in neuropeptide quantification using narrow bore LC, combined with ESI-MS, may be highly beneficial.

Capillary Action↗
PubMed2005

Characterization of a monolithic immobilized trypsin microreactor with on-line coupling to ESI-MS.

The preparation and characterization of a miniaturized trypsin reactor using on-line coupling with an ESI-TOF mass spectrometer are described. L-1-Tosylamido-2-phenylethyl chloromethyl ketone-trypsin was covalently immobilized on poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith prepared in a 75 microm ID fused silica capillary resulting in a bioreactor with high local concentration of the proteolytic enzyme. Covalent immobilization of trypsin on this support was performed using the epoxide functional groups in either a one- or a multistep reaction. For on-line protein digestion-MS analysis the bioreactor was coupled with the mass spectrometer using a liquid junction microelectrospray interface. The performance of the reactor was tested using an on-line flow through the system with flow rates of 50-300 nL/min. The resulting protein consumption was in the atto- to low femtomole range. Proteolytic activity was characterized in a wide range of conditions with respect to the flow rate, pH, and temperature. Complete protein digestion was achieved in less than 30 s at 25 degrees C with the sequence coverage of 80% (cytochrome c), which is comparable to 3 h digestion in solution at 37 degrees C. Besides the good performance at laboratory temperature, the immobilized trypsin in the bioreactor also performed well at lower pH compared to the standard in-solution protocols.

Capillary Action↗
PubMed2001

Determination of 4-hydroxytamoxifen in mouse plasma in the pg/mL range by gradient capillary liquid chromatography/tandem mass spectrometry.

Capillary high-performance liquid chromatography (HPLC; 300 microm i.d.) coupled to tandem mass spectrometry has been used to determine the concentration of 4-hydroxytamoxifen in mouse plasma in the pg/mL range following the administration of Tamoxifen. A limit of quantification (LOQ) of 100 pg/mL was achieved using only 25 microL of plasma. The on-column sensitivity was determined to be 100 fg. The column performance was determined isocratically before and after the assay and showed only a 15% reduction in performance after 70 injections of plasma extract. No significant peak band broadening was observed due to the mass spectrometer interface using a standard TurboIonspray source.

Administration, Oral↗
PubMed1994

Molecular entrapment of small molecules within the interior of horse spleen ferritin.

A procedure for trapping small molecules inside the interior of horse spleen ferritin (HoSF) and methods for characterizing HoSF and its small entrapped molecules are described. HoSF is first dissociated into subunits by adjustment to pH 2 in the presence of the small molecules to be trapped. The pH of the dissociated HoSF is then increased to 7 at which time the dissociated subunits reassemble reforming the 24-mer HoSF, thereby trapping solvent within its interior. HoSF is then separated from unbound molecules by dialysis, ultrafiltration, and/or ammonium sulfate precipitation. Sephadex G-25 and DEAE chromatographic methods were also used to separate HoSF from unbound small molecules. Capillary electrophoresis (CE) was used to demonstrate the association of small molecules with HoSF after the pH-induced unfolding-refolding process. The pH indicator neutral red was clearly associated with HoSF and presumed trapped within the ferritin interior. Acid/base titrations suggested that the trapped indicator had a different pKa than the free indicator, a result which indicates that the ferritin interior is different than the external solution. The utility of using trapped molecules for gaining information on ferritin function is proposed and discussed.

Ammonium Sulfate↗
PubMed1993

Blotting membrane micropreparation in capillary electrophoresis to evaluate enzyme purity and activity.

A micropreparation system for proteins and peptides analyzed by capillary electrophoresis that uses a blotting membrane and an improved micro-test-plate was developed. The system is useful for enzyme diagnosis and purification. In the system, enzyme is collected in a concentrated spot on these membranes, and the activities are measured enzymatically to evaluate the resolution of the system.

Capillary Action↗
PubMed1993

Automated ribosomal DNA fingerprinting by capillary electrophoresis of PCR products.

Capillary electrophoresis (CE) provides a rapid and automated technique for the analysis of subnanogram amounts of DNA fragments generated by the polymerase chain reaction (PCR). Here we describe the implementation of size-selective CE for DNA profiling and restriction fragment length polymorphism analysis of amplified polymorphic spacers of ribosomal DNA from fungi. Separations of unpurified and isopropanol-precipitated PCR-amplified DNA fragments in the size range of 20-1600 base pairs were achieved in less than 20 min with the use of hydroxypropyl methylcellulose as a sieving medium. The amplified internal transcribed spacer (ITS) and intergenic spacer (IGS) of RNA genes could be sized by coelectrophoresing a standard size ladder mixed with every sample, thereby eliminating errors in size estimation. This, together with the strictly controlled conditions of CE, permit the discrimination of amplified rDNA fragments differing only a few base pairs in length. Inter- and intraspecific variation in the size and number of restriction sites of the amplified rDNA spacers from the ectomycorrhizal basidiomycetes Laccaria laccata and Laccaria bicolor was observed and most strains could thus be reliably genotyped by PCR-CE. Multiple amplified IGS fragments of heterogeneous size were detected in several strains. This polymorphism is due to the occurrence of 5S rDNA subrepeats (i.e., multiple annealing of primer) within IGS. With CE, in contrast to slab gel electrophoresis, run times are short, the capillary can be reused, and full automation is feasible. Data acquisition and analysis are computer-controlled, which facilitates the locus identification and reduces error especially when large numbers of PCR products must be analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoanalysis↗
PubMed1994

A capillary electrophoresis method for studying apo, holo, recombinant, and subunit dissociated ferritins.

A capillary electrophoresis (CE) method is described for detecting and quantitating apo and holo ferritins from horse spleen (HoSF), rat liver (RLF), recombinant human light chain (rLF), recombinant human heavy chain (rHF), site-directed variants of human light chain, and Azotobacter vinelandii bacterial ferritin (AVBF). This procedure is carried out at pH 8.2, where the ferritin molecules are associated into their 24-mers. Protein mobilities as expressed as elution times were clearly resolved and could be used to distinguish one ferritin type from another, providing a means for detecting and quantitating various ferritin species in purified or partially purified states. Measurements of these and other ferritins were also conducted at pH 2.0, where dissociation into their respective subunits occurs. For HoSF and RLF, the individual L and H subunits were resolved and their relative concentrations were determined by integrating the areas of the elution peaks. HoSF gave 89.8% L and 10.2% H and RLF gave 70.7% L and 29.3% H, while rLF, rHF, and AVBF gave only a single subunit, all in agreement with reported values obtained by polyacrylamide gel electrophoresis. CE of HoSF, containing increasing amounts of iron in the interior, in general, showed that protein mobilities increased, reached a plateau, and then slowly decreased with increasing core size, although buffer effects altered this CE behavior to some extent. Such results indicate that species formed early during core formation have individual iron atoms present and differ from those formed later in which the oligomeric iron core has formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗