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Biomedical subjects

H Nohta

Publications and source records attributed to H Nohta.

At least 19 recordsLinked to original sources
PubMed1994

Fluorogenic reactions for biomedical chromatography.

A number of fluorogenic reactions, which have been used for HPLC detection systems by means of pre- and/or postcolumn derivatization, are surveyed with respect to both sensitivity and selectivity for the determination of biomedically important substances. For the derivatization of the substances, two types of fluorogenic reactions, fluorescence-generating and fluorescence-tagging, have been studied. The former are usable in most instances for both pre- and postcolumn derivatization methods, and the latter only for precolumn derivatization methods. HPLC methods utilizing the fluorogenic reactions allow analytes to be detected at picomole-subfemtomole levels. In the fluorescence-generating reactions, several fluorogenic reagents possessing two or more reactive sites in the molecule, which show molecular recognition for a variety of analytes, permit facile and reproducible detection in HPLC because there are fewer interferences from biological matrices.

Amines↗
PubMed1993

Determination of a cysteine protease inhibitor and its ethyl ester in mouse serum and muscle by liquid chromatography-mass spectrometry.

A liquid chromatographic-mass spectrometric method is described for the determination of a cysteine protease inhibitor (E64C) and its ethyl ester in mouse serum and muscle samples. The compounds in the sample, after deproteinization and solid-phase extraction, were separated by isocratic reversed-phase high-performance liquid chromatography and detected by on-line mass spectrometry. The use of an aqueous mobile phase containing methanol and 30 mM ammonium trichloroacetate provided abundant protonated molecular ions of the compounds in the atmospheric pressure chemical ionization interface of the detection system. The method permitted the quantitative determination of the inhibitors without internal standards in the biological matrices. The detection limits for the compounds, in the selected-ion monitoring mode, were 10-15 pmol on-column, at a signal-to-noise ratio of 5.

Animals↗
PubMed1992

High-performance liquid chromatographic determination of catecholamines and their precursor and metabolites in human urine and plasma by postcolumn derivatization involving chemical oxidation followed by fluorescence reaction.

A high-performance liquid chromatographic method for the determination of catecholamines and their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine, and L-DOPA), acidic compounds (3,4-dihydroxyphenylacetic acid, vanillyl-mandelic acid, and homovanillic acid), and alcoholic compound [4-hydroxy-3-methoxyphenyl)ethylene glycol)] in human urine and plasma. Urine and plasma samples deproteinized with perchloric acid in the presence of isoproterenol and 3,4-dihydroxyphenylpropanoic acid (internal standards) are fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction). The compounds in each fraction are separated by an ion-pair reversed-phase chromatography on a TSK gel ODS-80TM with isocratic elution and on-line derivatized by periodate oxidation followed by a fluorescence reaction using meso-1,2-diphenylethylenediamine. The detection limits (S/N = 5) vary from 0.5 to 95 pmol/ml, depending on the compounds.

Adult↗
PubMed1992

Determination of 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine in rat plasma by high-performance liquid chromatography with precolumn fluorescence derivatization.

A high-performance liquid chromatographic method with precolumn fluorescence derivatization using 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzofu ran is described for the quantification of 2',3'-dideoxyinosine, a therapeutic drug for acquired immunodeficiency syndrome, and 2',3'-dideoxyadenosine, an anti-human-immunodeficiency-viral agent, in rat plasma. The dideoxyribonucleosides and 3'-deoxythymidine (internal standard) in rat plasma (0.1 ml) are cleaned up by a solid-phase extraction technique using an octadecyl silica (ODS) cartridge, Toyopak ODS M, and the dideoxyribonucleosides in the eluate are reacted with the reagent to produce the corresponding fluorescent esters. The esters are separated by chromatography on a reversed phase column, TSKgel ODS-80TM. The detection limits (signal-to-noise ratio = 3) for the dideoxyribonucleosides are 1.3-5.4 pmol on column. Plasma concentrations of 2',3'-dideoxyinosine after intra-jugular-venous administration to rat can be monitored by this method.

Animals↗
PubMed1992

Precolumn derivatization of nucleotides based on fluorescent carbamate formation of the sugar moieties in high-performance liquid chromatography.

The fluorescence derivatization of nucleotides with 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzo++ furan in the presence of sodium azide and the separation of the derivatives by high-performance liquid chromatography are described. The reagent reacts with 5'-terminal hydroxyl groups of nucleotides to produce the corresponding fluorescent carbamates. The derivatives of mono- and oligonucleotides are separated by chromatography on a reversed phase column (TSKgel ODS-80TM) and the derivatives of octa- and deca-nucleotides on a size exclusion column (TSKgel G3000SWXL). The detection limits (signal-to-noise ratio = 3) are 0.8-6.0 pmol on column. 5'Phosphorylated nucleotide also gives a fluorescent derivative after alkaline phosphatase-mediated dephosphorylation.

Base Sequence↗
PubMed1992

A fluorescent DNA probe prepared by the direct derivatization of the sugar moiety for hybridization assay.

The preparation of a fluorescent DNA probe based on the derivatization of the terminal hydroxyl group of the sugar moiety of a DNA primer and its applicability to the DNA hybridization assay are described. M13mp8 plasmid primer reacts with 2-(5-chlorocarbonyl-2-oxazolyl)-5,6-methylenedioxybenzofu ran in the presence of sodium azide to form the corresponding fluorescent probe, which can be used for the hybridization assay to the target DNA, M13mp8 plasmid vector. The detection limit of the DNA with the naked eye is 10 ng (approximately 300 fmol)/spot on filters for the hybridization assay.

Base Sequence↗
PubMed1990

Determination of pseudouridine in human urine and serum by high-performance liquid chromatography with post-column fluorescence derivatization.

A selective and sensitive method for the determination of pseudouridine in human urine and serum is described. The method is based on high-performance liquid chromatography with post-column fluorescence derivatization. Pseudouridine and 5-fluorouridine (internal standard) in a 10-fold diluted urine sample or a deproteinized serum sample are separated on a reversed-phase column (TSK gel ODS-80) with isocratic elution and successively subjected to derivatization involving periodate oxidation followed by fluorescence reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine. The detection limit for pseudouridine is 4 pmol in a 100-microliters injection volume.

Chromatography, High Pressure Liquid↗
PubMed1990

1,2-Diarylethylenediamines as pre-column fluorescence derivatization reagents in high-performance liquid chromatographic determination of catecholamines in urine and plasma.

Meso- and dl-1,2-diarylethylenediamines (14 species) were evaluated for pre-column fluorescence derivatization reagents in the high-performance liquid chromatographic determination of catecholamines (norepinephrine, epinephrine and dopamine) in human urine and plasma. Of the compounds, meso-1,2-bis(4-methoxyphenyl)ethylenediamine was most preferable for all the catecholamines in terms of sensitivity and selectivity. The detection limit for each catecholamine is approximately 0.5 fmol in a 50-microliters injection volume.

Catecholamines↗
PubMed1990

Simultaneous determination of 2-deoxy-D-glucose and D-glucose in rat serum by high-performance liquid chromatography with post-column fluorescence derivatization.

A simple high-performance liquid chromatographic method for the determination of 2-deoxy-D-glucose and D-glucose in rat serum is described; this method is based on a post-column fluorescence derivatization. The sugars are automatically converted into fluorescent derivatives by reaction with meso-1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium after their separation on a strong anion exchanger column (TSK gel Sugar AXG). The detection limits (S/N = 3) for 2-deoxy-D-glucose and D-glucose in rat serum are 0.52 and 0.56 nmol/ml, respectively.

Animals↗
PubMed1989

Measurement of catecholamines, their precursor and metabolites in human urine and plasma by solid-phase extraction followed by high-performance liquid chromatography with fluorescence derivatization.

A high-performance liquid chromatographic method is described for the determination in human urine and plasma of catecholamines, their precursor and metabolites [amino compounds (norepinephrine, epinephrine, dopamine, normetanephrine, metanephrine, 3-methoxytyramine and L-DOPA), acidic compounds (3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, vanillylmandelic acid and homovanillic acid) and alcoholic compounds (3,4-dihydroxyphenylethyleneglycol and 4-hydroxy-3-methoxyphenylethyleneglycol)]. Urine (0.5 ml) containing 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid (internal standards) is deproteinized with perchloric acid, and the resulting solution is fractionated by solid-phase extraction on a strong cation-exchange resin cartridge (Toyopak IC-SP S) into two fractions (amine fraction and acid-alcohol fraction), which include 3,4-dihydroxybenzylamine and 4-hydroxy-3-methoxycinnamic acid, respectively. Plasma (0.7 ml) is deproteinized in the presence of 3,4-dihydroxybenzylamine (internal standard) in the same manner, and the resulting solution is directly used as an acid-alcohol fraction, while an amine fraction is obtained as for urine. Each fraction is subjected to the previously established ion-pair reversed-phase chromatography with post-column derivatization involving coulometric oxidation followed by fluorescence reaction with 1,2-diphenylethylenediamine. The detection limits, at a signal-to-noise ratio of 5, of the compounds measured in urine are 300 pmol/ml for the two mandelic acids, 2-7 pmol/ml for the other acidic and alcoholic compounds, 12 pmol/ml for L-DOPA and 0.6-2 pmol/ml for the other amino compounds; the corresponding values for plasma samples are 80, 0.5-3, 10 and 0.6-3 pmol/ml, respectively.

Catecholamines↗
PubMed1989

Simultaneous high-performance liquid chromatographic determination of catecholamine-related compounds by post-column derivatization involving coulometric oxidation followed by fluorescence reaction.

A highly selective and sensitive high-performance liquid chromatographic method for the determination of catecholamines (norepinephrine, epinephrine and dopamine) and related compounds (L-DOPA, normetanephrine, metanephrine, 3-methoxytyramine, 3,4-dihydroxymandelic acid, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid, 3,4-dihydroxyphenylethylene glycol, 4-hydroxy-3-methoxyphenylethylene glycol and 4-hydroxy-3-methoxyphenylethanol) with a post-column technique involving coulometric oxidation followed by fluorescence derivatization is described. These compounds, 3,4-dihydroxybenzylamine and ferulic acid are separated within 35 min by ion-pair reversed-phase chromatography using acidic buffers (pH 3.1) with methanol-acetonitrile (3:2, v/v) gradient elution, and then oxidized by a commercial coulometric detector to the corresponding o-quinones, which are converted into fluorescent derivatives by reaction with 1,2-diphenylethylenediamine. The detection limits (signal-to-noise ratio = 3) on-column are 1.5-4 pmol for the two mandelic acids, 600 fmol for L-DOPA and 20-70 fmol for the others.

Catecholamines↗
PubMed1987

Assay for dopamine beta-hydroxylase in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection.

A highly sensitive assay for dopamine beta-hydroxylase activity in human plasma and rat serum by high-performance liquid chromatography with fluorimetric detection is described. Norepinephrine, formed enzymatically from the substrate dopamine, and epinephrine (internal standard), after clean-up with a cation-exchange cartridge (Toyopak IC-SP M), are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-80TM. The detection limit for norepinephrine formed enzymatically is 5 pmol per assay tube.

Adult↗
PubMed1987

Assay for tyrosine hydroxylase by high-performance liquid chromatography with fluorescence detection.

A sensitive assay method for tyrosine hydroxylase in rat brain and adrenal medulla by high-performance liquid chromatography with fluorescence detection is described. L-DOPA formed enzymatically from the substrate L-tyrosine and alpha-methyldopa (internal standard), after clean-up with small cartridges of an activated alumina and a cation exchanger, Toyopak IC-SP M, are converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on TSK gel ODS-120T. The detection limit for L-DOPA formed enzymatically is 2 pmol per assay tube.

Adrenal Medulla↗
PubMed1987

Determination of free and total catecholamines in human urine by HPLC with fluorescence detection.

A simple and highly sensitive method for the determination of free and total (free + conjugated) catecholamines (norepinephrine, epinephrine and dopamine) in human urine is described which employs HPLC with fluorescence detection. Conjugated catecholamines (sulfate form) are hydrolyzed by a sulfatase-mediated reaction to the corresponding free amines. After cation exchange chromatography on a Toyopak IC-SP S cartridge, catecholamines and isoproterenol (internal standard) in urine samples were converted into the corresponding fluorescent compounds by reaction with 1,2-diphenylethylenediamine. These compounds were separated within 8 min on a reversed phase column with isocratic elution using a mixture of water, methanol and acetonitrile containing a Tris-hydrochloric acid buffer (pH 7.0). The detection limit for each catecholamine is ca 2 fmol per 100 microL injection volume.

Adult↗
PubMed1986

Occurrence of aromatic L-amino acid decarboxylase in human plasma and its assay by high-performance liquid chromatography with fluorescence detection.

A high-performance liquid chromatographic method using fluorescence detection for assessing the activity of aromatic L-amino acid decarboxylase in human plasma is described. Dopamine, formed enzymatically from L-DOPA, and isoproterenol (internal standard) are chromatographed on a small ion-exchange cartridge (Toyopak SP) and derivatized with 1,2-diphenylethylenediamine. The derivatives are separated by reversed-phase chromatography on an Ultrasphere ODS column. The detection limit for dopamine formed enzymatically is 0.6 pmol per 500 microliter of enzyme reaction mixture. Aromatic L-amino acid decarboxylase in human plasma is very similar to that in rat kidney, with respect to optimum conditions for the enzyme reaction and gel chromatographic behaviour.

Adult↗