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Biomedical subjects

A Shibukawa

Publications and source records attributed to A Shibukawa.

At least 37 records · Page 2Linked to original sources
PubMed1995

Chemical stability of polyacrylamide-coating on fused silica capillary.

The chemical stability of polyacrylamide coatings on a fused silica capillary for capillary electrophoresis was investigated with respect to temperature of capillary and pH as well as salt concentration of running buffer solution by evaluating their effects on electroosmotic mobility. The capillary covalently bonded to linear polyacrylamide through Si-C linkage was stable in buffer solutions of pH 2.3-8.0 at 30 degrees C for 30 days, whereas the capillary bonded through Si-O-Si-C linkage was damaged at pH above 4.6. The electroosmotic mobility observed in both capillaries increased with increasing temperature, though the degree of increase was smaller in the Si-C linked capillary than in the Si-O-Si-C linked capillary. The increase in the buffer concentration resulted in increased electroosmotic mobility in the Si-O-Si-C linked capillary, but no effect was observed for the Si-C linked capillary.

Acrylic Resins↗
PubMed1995

Michaelis-Menten analysis of immobilized enzyme by affinity capillary electrophoresis.

Michaelis constant of enzymatic reaction was evaluated by affinity capillary electrophoresis using beta-galactosidase as a model enzyme and o- and p-isomers of nitrophenyl-beta-galactoside as substrates. The enzyme was immobilized on the inner surface of a fused-silica capillary by the covalent bonding through a bridging group, and the substrates were introduced into the capillary. The reaction products migrated electrophoretically toward the detection side (anodic side), while the unreacted substrates moved toward the injection side (cathodic side) on a slow electroosmotic flow generated by the weak negative charge of the immobilized enzyme. The initial velocity of the enzymatic reaction was estimated from the peak height of the product, and the Michaelis constant was calculated according to Lineweaver-Burk equation. The results (Km, 2.34 mM for o-isomer and 1.09 mM for p-isomer) were reproducible (RSD < 11.8%, n = 5). Although the estimated Michaelis constants were larger than the reported values measured in homogeneous solution, the ratio of the Michaelis constants of o-/p-isomers was in good agreement with the literature value. The present method required as low as a few microgram amount of enzyme and nanogram amount of substrate which is far smaller than those required in a conventional affinity HPLC.

Amides↗
PubMed1994

High-performance capillary electrophoresis of SDS-proteins using pullulan solution as separation matrix.

Sodium dodecyl sulphate (SDS) capillary electrophoresis using pullulan solution as a separation matrix was developed for the separation and molecular mass determination of proteins. The silanol functions on the inner surface of a fused-silica capillary were deactivated by coating with linear polyacrylamide through Si-C linkages, into which the pullulan solution was filled. The stability of the coating was examined by exposure to an alkaline buffer solution (pH 9.2) for up to 30 days. Compared with conventional coatings with linear polyacrylamide through siloxane linkages, the present capillary was more stable even under alkaline conditions and markedly reduced electroosmotic flow. Thus, polymer solutions of low viscosity such as pullulan solution could be stabilized in the capillary, resulting in a prolonged life-span of the capillary and improved reproducibility of separations. An excellent linear relationship was obtained between the mobility and the logarithm of the molecular mass of SDS-proteins. The relative standard deviation of migration times was below 0.5% when the pullulan solution was refilled in each analysis (n = 10). The calibration plots of the integrated peak areas at 214 nm vs. concentration of standard proteins were linear in the range 5 micrograms/ml-0.1 mg/ml.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

High-performance capillary electrophoresis/frontal analysis for the study of protein binding of a basic drug.

A simple high-performance capillary electrophoresis (HPCE) method based on the principle of frontal analysis was applied to the determination of the concentration of unbound basic drug in protein binding equilibrium. A small volume of sample solution (approximately 80 nL) containing 113-340 microM of verapamil (VER) and 100-550 microM of human serum albumin was introduced into the fused silica capillary (effective length, 22 cm; 50-microns i.d.) by suction. Because the silanol groups on the inner surface of the capillary were bound with linear polyacrylamide through Si-C bonds, electroosmotic flow was not generated even at pH 7.4 with an applied voltage of +10 kV. The unbound drug bearing positive charge migrated electrophoretically from the drug-protein mixed zone toward the detection end, whereas human serum albumin did not co-migrate because of its negative charge. The bound drug migrated after it was released from the protein. As a result of an 80-nL injection of the sample solution, VER was eluted as a zonal peak with a plateau region. The VER concentration calculated from the plateau height agreed well with the unbound VER concentration determined by the conventional ultrafiltration-HPLC method, with good reproducibility (CV, < 6.23%, n = 15). The present HPCE/FA system was applied to the Scatchard analysis of VER and alpha 1-acid glycoprotein binding, and the estimated binding parameters agreed well with literature values.

Blood Proteins↗
PubMed1994

Determination of the unbound concentration of hydrophobic drugs in albumin solutions by high-performance frontal analysis using a diol-silica column.

High-performance frontal analysis (HPFA) using diol-silica column allows the determination of the unbound concentration of hydrophobic drugs under protein binding equilibrium, which is often difficult by the conventional methods. After the direct injection of a drug-protein mixed solution onto a diol-silica column, the drug is eluted as a zonal peak containing a plateau region under a mild mobile phase condition (phosphate buffer, pH 7.4, ionic strength = 0.17). The unbound drug concentration was determined as the drug concentration in the plateau region. The reliability of this method was confirmed by comparison with the results obtained by the conventional ultrafiltration-HPLC method. The versatility of the HPFA method using a diol-silica column was demonstrated by developing a novel on-line HPFA-chiral HPLC system for a simple and easy determination of the unbound concentration of nilvadipine (NV) enantiomers. The direct injection of 1.33 mL of a sample solution containing 20 nM-1 microM NV and 550 microM HSA gave the plateau region due to unbound NV, which was heart-cut and transferred on-line into a preconcentration and chiral HPLC separation system. As low as a few hundred picomolar level of unbound NV enantiomers was determined by use of UV detection (at 244 nm). The binding between NV and HSA is enantioselective; (R)-NV binds more strongly than (S)-NV.

Chromatography, High Pressure Liquid↗
PubMed1993

Sodium dodecyl sulfate-polyacrylamide solution-filled capillary electrophoresis of proteins using stable linear polyacrylamide-coated capillary.

Sodium dodecyl sulfate (SDS)-linear polyacrylamide solution-filled capillary electrophoresis using a stable coated fused silica capillary has been developed for the separation and molecular weight determination of proteins. A fused silica capillary was coated with linear polyacrylamide through Si-C bond which suppressed the electro-osmotic flow and reduced the adsorption of proteins. Acrylamide solution containing SDS was filled into the coated capillary and polymerized. Compared with a conventional fused silica capillary coated with 3-methacryloxypropyltrimethoxysilane through siloxane linkage (Si-O-Si-C), the developed capillary was much more stable, even in the alkaline condition. Consequently, the durability of the polymer matrices and the reproducibility of separation were markedly improved. An excellent linear relationship was obtained between the mobility and the logarithm of the protein molecular weight. The relative standard deviation of migration times was below 1%.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Direct injection analysis of atenolol enantiomers in plasma using an achiral/chiral coupled column HPLC system.

A novel HPLC system was developed with an achiral/chiral coupled column for the direct injection analysis of atenolol (AT) enantiomers in plasma. The system consists of a size-exclusion column, an ODS silica column and a newly developed beta-cyclodextrin perphenylcarbamate(ph-beta-CD)-bonded silica column connected in a series via two switching valves. The neat plasma sample was directly injected onto the size-exclusion column, and the deproteinized fraction of AT enantiomers was concentrated on the ODS silica column. The enantiomers were then transferred and separated mutually on the ph-beta-CD silica column. The calibration line for each of the AT enantiomers was linear in the range of plasma concentration of 10-200 ng/ml (r > 0.9997) with good reproducibility (CV < 9.7%, n = 20). The recoveries from plasma were almost complete (> 97.4%) for both enantiomers. One analysis finished within 30 min. The developed system was applied to the enantioselective determination of plasma concentration-time curve of AT after the oral administration of racemic AT to a healthy volunteer.

Atenolol↗
PubMed1992

Simultaneous direct determination of the enantiomers of verapamil and norverapamil in plasma using a derivatized amylose high-performance liquid chromatographic chiral stationary phase.

Chiralpak AD is a commercially available high-performance liquid chromatographic column containing a chiral stationary phase composed of 3,5-dimethylphenylcarbamate-derivatized amylose coated on silica. This column was applied to the assay for the plasma concentrations of the enantiomers of verapamil and its major metabolite norverapamil. After the extraction from plasma, the analytes were separated on a diol silica column (LiChrocart DIOL) and Chiralpak AD column which were connected in series in this order and detected using a fluorescence detector (excitation at 272 nm, emission at 317 nm). The enantiomers of verapamil and norverapamil were separated from each other and other metabolites using a mobile phase of hexane-isopropanol-ethanol (85:7.5:7.5, v/v/v) containing 1.0% triethylamine. The calibration curves were linear (R greater than 0.9989) in the plasma concentration range 2.5-100 ng/ml for verapamil enantiomers and 5.0-100 ng/ml for norverapamil enantiomers. The intra-day and inter-day reproducibility tests showed good reproducibilities; coefficients of variation of each enantiomer were less than 14.9% at the lowest concentration and less than 2.0% at the highest concentration. The effect of organic modifier content and column temperature upon the retention and the enantioseparation were also discussed.

Amylose↗
PubMed1992

High-performance frontal analysis-high-performance liquid chromatographic system for stereoselective determination of unbound ketoprofen enantiomers in plasma after direct sample injection.

An on-line, high-performance frontal analysis (HPFA)-high-performance liquid chromatographic system was developed for the enantioselective determination of a low level of unbound ketoprofen (KP) that is in equilibrium with KP that is bound to protein. The system consists of three subsystems (HPFA system, preconcentration system, and chiral separation system) connected in series in the stated order via column-switching valves. When either a 300-microL portion of buffer solution containing 300 or 550 microM human serum albumin and 100 or 300 microM racemic KP or a 300-microL portion of human plasma containing 12.5-100 microM racemic KP was directly injected onto the HPFA column with the mobile phase at a low flow rate, KP was separated from proteins and eluted as a zonal peak with a plateau. The KP concentration in the eluant of the plateau region was the same as the unbound-KP concentration that was in equilibrium with protein-bound KP in the initial sample solution. A 1-mL portion of the eluant of the plateau region was switched to the preconcentration system, where KP was adsorbed and condensed on an octadecylsilyl silica (ODS) column. The adsorbed KP was eluted out of the ODS column and transferred to the chiral separation system, via another switching valve, where the enantiomers of unbound KP were separated and determined. The results agree well with those obtained by the conventional ultrafiltration method.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromatography, High Pressure Liquid↗
PubMed1992

Direct injection analysis of carbamazepine and its active 10,11-epoxide metabolite in plasma by use of a semipermeable surface (SPS) silica column in LC.

A semipermeable surface (SPS) silica column was applied for the simultaneous determination of carbamazepine (CBZ) and its active 10,11-epoxide metabolite (EPO) in plasma following direct injection in LC. The SPS packing material consists of an ODS ligand as the hydrophobic inner phase and a polyoxyethylene network as the hydrophilic outer phase. When a 5-microliters portion of intact plasma was injected onto the column using a mobile phase of phosphate buffer (pH 7.1, ionic strength 0.1)-acetonitrile (4:1, v/v), the plasma proteins were size-excluded, whereas the drug and its metabolite were retained and separated both from each other and from other commonly co-administered drugs such as phenobarbital (PB) and phenytoin (DPH). The calibration graphs (peak area vs concentration) of CBZ, EPO and PB were linear over the therapeutic range of plasma concentration (r greater than 0.998) with good relative standard deviations (RSD less than 3.98%, n = 5). The recoveries from plasma were almost complete (greater than 96.6%). The analysis time was 17 min. The method as developed was applied in studies on the time course of plasma concentrations of unchanged CBZ and EPO after i.v. administration of CBZ to the rat.

Calibration↗
PubMed1990

Stereoselective determination of free warfarin concentration in protein binding equilibrium using direct sample injection and an on-line liquid chromatographic system.

A new on-line high-performance liquid chromatography system was developed for the stereoselective determination of free drug concentration in drug-protein binding equilibrium. When a 40-300-microL portion of a sample solution containing 50-200 microM racemic warfarin (Wf) and 100-550 microM human serum albumin was directly injected into the internal-surface reversed-phase silica column, Wf gave a trapezoidal peak exhibiting a plateau region. The concentration in the plateau region was equal to the free Wf concentration in the initial sample solution. By the delivery of a portion (90 microL) of the eluent in the plateau region into the chiral separation column (Chiral AGP column) by column switching, the free concentrations of respective enantiomers of Wf were determined. The results agreed well with those obtained by the conventional ultrafiltration method. The precision was also confirmed by the within-run and day-to-day reproducibilities (coefficient of variation less than or equal to 3.05%, n = 5). The present method is simple and rapid and four sample solutions can be analyzed within 1 h without pretreatment.

Chromatography, High Pressure Liquid↗
PubMed1990

Direct injection analysis of ketoprofen enantiomers in plasma using column-switching high-performance liquid chromatography system.

A high-performance liquid chromatography system was developed for the stereoselective determination of ketoprofen enantiomers in human plasma following direct sample injection. The system comprised of a pretreatment column and a chiral separation column connected in a series via a switching valve. When a 200 microliter portion of human plasma containing a therapeutic level of ketoprofen was directly applied to the system, ketoprofen was adsorbed in the pretreatment column, while plasma proteins were excluded. After the elution of proteins from the pretreatment column, the valve was switched and ketoprofen was desorbed and transferred to the chiral separation column where the enantiomers were separated and determined by ultraviolet-absorption. The mobile phase conditions for the pretreatment and chiral separation were optimized, which enabled rapid and complete recovery followed by satisfactory separation of the enantiomers. The calibration line for each enantiomer showed good linearity in the range of 0.25-5 micrograms/ml with a detection limit of 0.02 micrograms/ml (signal to noise ratio (S/N) greater than 3), which was sufficient for practical demands. The precision test indicated that the coefficient of variation for five repeated determinations of (-) ketoprofen was 5.4% at 0.1 microgram/ml and 1.4% at 1 microgram/ml.

Chromatography, High Pressure Liquid↗
PubMed1990

Simultaneous determination of the free and total carbamazepine concentrations in human plasma by high-performance frontal analysis using an internal-surface reversed-phase silica support.

High-performance frontal analysis (HPFA) was applied to simultaneous determinations of the free and total carbamazepine (CBZ) concentrations in human plasma. When 1.5ml of human plasma containing CBZ at a clinical therapeutic level (free fraction, about 30%) was directly injected into an internal-surface reversed-phase silica column, the CBZ was separated from the plasma blank and was eluted as a zonal peak with a plateau height corresponding to the free CBZ concentration in protein binding equilibrium. Slow and continuous introduction of the plasma sample and the use of titanium filters permitted us to inject the sample repeatedly while avoiding a rapid increase in column pressure. The free and total CBZ concentrations were determined simultaneously from the peak height at the plateau region and the area of the CBZ peak, respectively. The within-run and day-to-day reproducibilities were satisfactory (C.V. less than or equal to 1.63%, n = 5).

Carbamazepine↗
PubMed1989

Effect of protein binding on high performance liquid chromatography analysis of drugs with an internal-surface reversed-phase silica column.

The effect of protein binding on the high performance liquid chromatography (HPLC) elution profile of drugs injected directly onto an internal-surface reversed-phase (ISRP) silica column was investigated. When a relatively large volume (greater than or equal to 100 microliters) of the sample solution containing warfarin and bovine serum albumin (BSA) was applied directly onto the ISRP column and elution was carried out with the mobile phase containing an organic modifier, two distinct peaks, both due to warfarin, appeared separated from the protein peak. The peak splitting was not observed in the case of antipyrine-BSA mixed solution, where the protein binding is weak. It was found that the drug bound at the strong-binding site on the BSA molecule was released slowly during the process of chromatography and was eluted as the first peak with a shorter retention time, while the drug bound at the weak-binding site was released quickly, and was eluted as the second peak together with the free drug. The warfarin-BSA interaction at the strong-binding site was evaluated, under minimum influence from the other binding site, from the warfarin concentration obtained from the first peak and the free warfarin concentration determined by the ultrafiltration method.

Animals↗
PubMed1989

Development of effective cross-linking method for bioactive substance--enzyme immobilization using glutaraldehyde oligomers.

When a 10% aqueous solution of glutaraldehyde (GA) was alkalized to pH 8.5 in borate buffer solution and heated at 60 degrees C, the ultraviolet spectrum of GA solution showed two distinct absorption maxima. The one at 280 nm with a weak absorbance ascribable to the C = O bond in the aldehyde group shifted to near 300 nm after 50 min with a slight increase in its intensity. Another maximum at 235 nm with a strong absorbance was ascribable to the C = C bond of the alpha,beta-unsaturated aldehyde group which was formed by aldol condensation reaction of GA monomer, and its absorbance increased markedly with increasing reaction time. The high performance liquid chromatography (HPLC) analysis with detection at 235 nm indicated that several GA oligomers were formed by the alkali treatment and their concentrations increased. The cross-linking ability of these oligomers was examined by immobilizing enzymes (alcohol dehydrogenase (ADH), glutamate dehydrogenase (GLDH] to an aminated polymer gel matrix by reaction with the treated GA solution. The enzyme activities increased with increasing concentration of GA oligomers. Then, the GA oligomers were isolated and used as the cross-linking agent. The activities of ADH and GLDH were 4-fold and 13-fold higher, respectively, than those obtained by using untreated GA solution, while the total amounts of immobilized enzymes were almost unchanged. These results suggest that GA oligomers may act as cross-linkers in a manner different from the generally accepted Schiff base formation reaction; a possible mechanism may involve addition reaction of an amino group to the double bond in the aldol condensate of GA.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗
PubMed1989

Determination of free drug in protein binding equilibrium by high-performance frontal analysis using internal-surface reversed-phase silica support.

An internal-surface reversed-phase silica column was employed in the frontal analysis method to determine free drug concentration in warfarin (Wf)-bovine serum albumin (BSA) mixed solution and in indomethacin(Im)-BSA mixed solution. When a 4-ml portion of aqueous solution containing 2-30 microM Im and 28 microM BSA and a 10-ml portion of aqueous solution containing 0.5-175 microM Wf and 28 microM BSA were applied, the elution curves reached a plateau level corresponding to both free drug and drug-BSA complex (beta-plateau), followed by another plateau due to free drug alone (gamma-plateau). The drug concentration at the gamma-plateau agreed well with the free drug concentration determined after ultrafiltration of the same solution. The gamma-plateau was observed even when the applied volume was reduced to a level which was insufficient to produce the beta-plateau. The injection of a 400-microliters portion of the 0.5-100 microM Im and 28 microM BSA mixed solution and a 90-microliters portion of 50 microM Im and 550 microM BSA mixed solution onto the ISRP silica column gave a clear gamma-plateau. Compared to the conventional frontal analysis, the present method can determine a wide range of drug levels with much smaller injection volume. This method is applicable to plasma. By a single frontal analysis, both free and total concentrations of carbamazepine in plasma were determined simultaneously.

Animals↗
PubMed1989

Electrokinetic chromatography for drug analysis. Separation and determination of cefpiramide in human plasma.

Electrokinetic chromatography using a fused silica capillary and sodium dodecyl sulfate (SDS) solution has been applied to the separation and determination of cefpiramide (CPM) in human plasma with the use of antipyrine (AP) as an internal standard. A plasma sample was introduced into the capillary by siphoning. The calibration plot for CPM in plasma sample showed good linearity in the concentration range over 10 to 300 micrograms/ml. This method has advantages over usual high performance liquid chromatography (HPLC) in that it needs only a very small volume (less than 10 nl) of plasma without pretreatment, and an extremely high separation efficiency (10 times or much higher plate number than usual HPLC) is obtained. The addition of SDS to the supporting electrolyte solution enabled (1) rapid release of protein-bound drug which allowed the total concentration to be determined, (2) reproducible results to be obtained by suppressing adsorption of protein onto the fused silica capillary and (3) rapid separation of drug from proteins by selective retardation of protein peaks.

Cephalosporins↗