Tokuichiro Seki

Simple method for the simultaneous determination of noradrenaline, dopamine and serotonin by stepwise elution from a short column of weak cation-exchange resin

A simple method for the simultaneous determination of noradrenaline, dopamine and serotonin using a short column of Amberlite CG-50 is described. Noradrenaline and dopamine were eluted from the column with phosphate buffers containing 1.5 and 4.0% boric acid, respectively, and then serotonin was eluted with 1.0 N HCl. Catecholamines were determined by a modification of the ethylenediamine condensation method using potassium ferricyanide as oxidant and isobutanol for extraction of the fluorophores. Serotonin was measured by the acidic o-phthalaldehyde method. The method was applied to the simultaneous determination of noradrenaline, dopamine and serotonin in discrete regions of rat brain.

Modified ethylenediamine condensation method and its application in the analysis of catecholamines by ion-exchange chromatography

Abstract A modified ethylenediamine condensation method is described in which catecholamines dissolved in a borate buffer are convertedd into fluorescent products by oxidation with hexacyanoferrate(III) in the presence of ethylenediamine under alkaline conditions. The method was applied successfully in the fluorimetric determination of catecholamines eluted from a column of weakly acidic ion-exchange resin with a mixed buffer of pH 6.3 containing 0.35 M boric acid and 0.002 M disodium ethylenediaminetetraacetate. An analysis of human urine for catecholamines is described.

Simultaneous determination of uric acid and creatinine in biological fluids by column-switching liquid chromatography with ultraviolet detection

A column-switching liquid chromatographic method for the simultaneous determination of uric acid and creatinine in human serum and urine was developed. Creatinine and uric acid were separated by size-exclusion chromatography on a hydrophilic gel column (C1) and creatinine eluted from C1 was separated from proteins by filtration through a longer hydrophilic gel column (C2). The creatinine fraction eluted from C2 was transferred to a weakly acidic cation-exchange column (C3) and then to a strongly acidic cation-exchange column (C4). Uric acid eluted from C1 after creatinine was transferred to an anion-exchange column (C5) and then to a hydrophilic gel column (C6). The mobile phase was a mixed buffer of pH 5.1 (propionic acid-succinic acid-NaOH, 60:15:60 mmol/l in water). Diluted serum and urine could be injected onto C1, and C1 was backflushed after the transfer of uric acid from C1 to C5. Creatinine and uric acid in the eluate were determined by measuring their ultraviolet absorption at 234 and 290 nm, respectively. The recovery of uric acid and creatinine added to diluted serum (20-fold dilution, concentration 20 and 5 mumol/l, respectively) was 98.9 +/- 0.56% and 100.9 +/- 1.29%, respectively. The recovery of uric acid and creatinine added to diluted urine (100-fold dilution, concentration 50 and 100 mumol/l, respectively) was 99.4 +/- 0.72% and 98.7 +/- 1.45%, respectively (mean +/- R.S.D., n = 6).

Estimation of catecholamines in human plasma by ion-exchange chromatography coupled with fluorimetry

Estimation of catecholamines in human plasma was made by ion-exchange chromatography coupled with fluorimetry. Catecholamines in deproteinized plasma were adsorbed onto Amberlite CG-50 (pH 6.5, buffered with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid. The catecholamine fraction was separated further on a column of Amberlite IRC-50 which was coupled with a device for the automated performance of the trihydroxyindole method (epinephrine and norepinephrine) or the 4-aminobenzoic acid-oxidation method (dopamine). One sample could be analysed within 25 min with either method. The lower detection limits were 0.02 ng for epinephrine and dopamine, and 0.04 ng for norepinephrine. Plasma catecholamine contents of healthy adults at rest were epinephrine 0.07 +/- 0.01 ng/ml (n = 19), norepinephrine 0.27 +/- 0.03 ng/ml (n = 19) and dopamine 0.22 +/- 0.03 ng/ml (n = 26). The procedure of adsorption and elution of the plasma catecholamines by ion-exchange resin was simple, the simplicity contributing to constant recovery. The catecholamine fraction could be analysed without evaporation of the eluate. The analytical column could be used for the analysis of more than 1000 samples before excessive back-pressure developed. Our method of continuous measurement of plasma catecholamine fulfils clinical requirements.

Chromatographic separation of derivatives of phenol on cation exchange resins

Abstract It was shown that three isomers of mononitrophenol and of dihydroxybenzene were separated on a column of Amberlite IR 112 or of Duolite C 25 using solvent A or C. Separation of polynitrophenols was successful on a column of Duolite C 25 using solvent B. Isomers of cresol were separated from each other on a column of Amberlite IR 112 using solvent E and m-cresol was separated from other cresols on a column of Duolite C 25 using solvent F. Hydroquinone, pyrocatechol, phenol, p-cresol and p-tert.-butylphenol could be separated on a column of partially esterified Amberlite IRC 50 using solvent D.

Determination of free catecholamines in human urine by direct injection of urine into a liquid chromatographic column-switching system with fluorimetric detection

An ion-exchange chromatographic method combined with ion exclusion was developed for the determination of free catecholamines in human urine. Catecholamines were separated by ion exclusion from most acidic and neutral impurities by filtration through an anion-exchange column with a hydrophilic matrix (Asahipak ES-502N) and the excluded catecholamines were separated by ion-exchange chromatography on a column of weakly acidic ion exchanger with a hydrophilic matrix (Asahipak ES-502C), connected in series to the Asahipak ES-502N column with a four-way automatic valve. A sodium succinate-borate buffer of pH 6.7 (0.035 mol of succinic acid, 0.0075 mol of borate and 0.5 mmol of ethylenediaminetetraacetate were dissolved in 1 kg of water and the pH of the solution was adjusted to 6.7 with sodium hydroxide) was used as the mobile phase, and the temperature of both columns was kept at 30 degrees C. The catecholamines in the eluate were determined fluorimetrically by post-column derivatization with glycylglycine. A diluted urine sample was injected directly onto the first column. The first column was back-flushed with the mobile phase for 52.5 min after the elution of the catecholamines from the first to the second column. Then the columns were washed with the mobile phase for 10 min in the normal direction before the next sample was injected into the first column. Samples could be analysed every 70 min and 5 pmol/ml of epinephrine, 5 pmol/ml of norepinephrine and 25 pmol/ml of dopamine in human urine could be determined.

Estimation of catecholamines by ion-exchange chromatography on Asahipak ES-502C, using glycylglycine as the post-derivatizing agent.

The estimation of catecholamines in human urine was carried out by ion-exchange chromatography on a column of a weakly acidic ion exchanger with an hydrophilic matrix. The catecholamines were first adsorbed onto Amberlite CG-50 (buffered at pH 6.5 with 0.4 M phosphate buffer) and selectively eluted by 0.66 M boric acid solution. They were then separated from impurities that responded to fluorometric detection by isocratic elution from a column of Asahipak ES-502C, a cross-linked vinyl alcohol copolymer with carboxymethyl groups, at 60 degrees C. The mobile phase was 0.05 M sodium succinate buffer pH 5.25 containing 0.015 M borate and 0.5 mM ethylenediaminetetraacetate. Isoproterenol was used as the internal standard; epinephrine, norepinephrine, isoproterenol and dopamine were eluted in this order. One sample could be analyzed every 35 min. The detection limits were 0.2 ng for epinephrine and norepinephrine, 0.6 ng for dopamine. The elution pattern was quite reproducible; the elution volumes of the catecholamines had not changed after 500 determinations.

Chromatographic separation of dansyl amino acids and dansyl amines on amberlite IRC-50.

Abstract A column chromatographic method for the separation of dansyl amino acids and dansyl amines is described in which Amberlite IRC-50 equilibrated with the eluent is used as the stationary phase and six solvent mixtures, which are composed of a buffer or 2 % acetic acid and tetrahydrofuran, methyl ethyl ketone and acetone, are used as eluents. Neutral and acidic dansyl derivatives were separated at low pH, and those having a positive charge or present as the dipolar ion at pH 5.60 were separated at high pH. Quantitative determination of histamine as its monodansyl derivative was successful, and an analysis for histamine in a rat liver extract is described.

Separation of ascorbic acid, dehydroascorbic acid, diketogulonic acid and glucose by isocratic elution from a column of a hydrophilic gel

High-performance liquid chromatography on an Asahipak GS-320 hydrophilic gel column with tartrate buffer (0.015 M, pH 3.0) containing 2 mM ethylenediaminetetraacetate and 0.05% beta-thiodiglycol as the eluent allowed the separation of glucose, diketogulonic acid, dehydroascorbic acid and ascorbic acid within 30 min. Fluorimetric monitoring of these compounds in the eluate with benzamidine at alkaline pH and at 90 degrees C in the presence of potassium sulphite allowed the determination of nanogram amounts of ascorbic acid, dehydroascorbic acid and diketogulonic acid. This method was applied to the determination of ascorbic acid in fruit juice.

Anaesthetic management with morphine in phaeochromocytoma

Morphine was used as the principal anaesthetic agent for five patients undergoing resection of phaeochromocytoma, and changes in plasma catecholamines were monitored. Phenoxygenzamine (0.5 mg.kg-1.day-1) was orally given one week before operation. After premedication with hyoscine (0.4 mg) and diazepam (20 mg) of meperidine (50 mg), anaesthesia was induced with an infusion of morphine (1 mg.kg-1) over 30 min and maintained with diazepam, nitrous oxide and oxygen. Pancuronium was used as the muscle relaxant. In three of the five patients, blood pressure transiently rose (ranging from 15 to 60 mg) following orolracheal intubation, but vasodilators were not required. In two of the three, plasma norepinephrine increases were 1.1 and 1.3 ng.ml-1. In the other two patients, whose blood pressure was stable, the change in plasma norepinephrine was 0.1 and 0.7ng.ml-1. After reaching a peak at the time of ligation of drainage vein from the tumour, plasma calecholmines rapidly decreased to the preoperative levels, but the blood pressure was well maintained in all cases. Ventricular arrhythmias did not occur despite the increased levels of endogenous catecholamines.These findings suggest that morphine can be used as an alternative anaesthetic agent during operation for phaeochromocytoma and that exaggerated pressor responses to morphine (when given slowly) seem not to occur in phaeochromocytoma.RésuméChez cinq malades, on a effectué une résection d’un phéochromocytome sous aneslhésie à la morphine en mesurant, durant l’intervention, les fluctuations des concentrations des catécholamines plasmatiques. Les malades ont été préparés â la phénoxybenzamine (0.5 mg.kg-1.jour-1) administrée par voie orale dans la semaine précédant l’intervention. A la suite d’une prémédication faite d’hyoscine (0.4 mg) et de diazépam (20 mg) ou de mépéridine (50 mg), on a induit l’anesthésiepar une infusion de sulfate de morphine (S mg.kg-1) sur une période de 30 minutes, complétée de diazépam, de proloxyde d’azote, d’oxygène et de bromure de pancuronium. Chez trois de ces cinq malades on a observé une augmentation transitoire de la pression artérielle de 15 à 60 mmHg au moment de l’intubation orotrachéale, mais cette augmentation n’a pas nécessité de vasodilatateurs. Chez deux de ces trois malades, on a observé une augmentation de la norépinéphrine plasmatique de 1.1 et 1.3 ng.ml-1respectivement. Chez les deux autres malades dont la pression artérielle est restée stable, les concentrations de norépinéphine plasmatique étaient respectivement de 0.1 et O7ng.ml-1. Une fois atteint le pic de concentration des catécholamines plasmatiques au moment de la ligature de la veine drainant la tumeur, les concentrations ont rapidement diminué au niveau préopératoire alors que la pression artérielle demeurait stable en tout temps. Aucune arythmie ventriculaire n’est apparue en dépit de ces variations des catécholamines endogènes. Ces constatations suggèrent que la morphine constitue un agent anesthésique acceptable pour la résection d’un phéochromocytome car les flambées hypertensives en réponse à la morphine administrée lentement ne semblent pus survenir en présence d’un phéochromocytome.