Shigeo Takeshima

Fluorometric determination of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography using enzyme reactors.

A selective and sensitive assay of hypoxanthine and xanthine in biological fluids by high-performance liquid chromatography coupled with immobilized-enzyme reactors was developed. The separations were achieved by reversed-phase liquid chromatography. Hydrogen peroxide produced from hypoxanthine and xanthine by immobilized xanthine oxidase was determined fluorometrically using immobilized peroxidase and p-hydroxyphenylacetic acid. Immobilized enzymes were prepared by intermolecular cross-linking to controlled-pore glass. Assay of allopurinol was also possible by the present method. The method was applied to serum and urine. The detection limits of hypoxanthine and xanthine were approximately 50 and 120 pg per injection, respectively.

Transport Behavior of Basic Amino Acids through an Organic Liquid Membrane System

Abstract The transport behavior of basic amino acids (BAA), such as arginine (Arg), histidine (His), and ornithine (Orn), through an organic liquid membrane system (LMS) was investigated. The LMS was composed of two aqueous phases (Phases I and II) separated by an organic phase of chloroform containing sodium di-(2-ethylhexyl) sulfosuccinate (Aerosol OT, AOT). The amount of BAA that moved from Phase I at pH 3 into the organic phase increased with increasing AOT concentration (2–10 mM). The relative amount of extracted BAA was in the following order: Arg > His > Orn. On the other hand, the release of BAA from the organic phase into Phase II at pH 10 did not depend upon their amount in the organic phase. Arg was difficult to release. The relative amount of released BAA was in the following order: Arg = His > Orn. BAA were extracted from Phase I at pH 5 into the organic phase containing 4 mM AOT because they exist as cationic species. Other amino acids possessing nonionic residues were untransportable under ...

High-performance liquid chromatography with chemiluminescence detection of serum levels of pre-column derivatized fluoropyrimidine compounds

7-(Diethylamino)-3-[4-[iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) and 4-(bromomethyl)-7-methoxycoumarin have been evaluated as fluoropyrimidine-derivatizing agents to be detected using peroxyoxalate chemiluminescence with high-performance liquid chromatography. The derivatization procedure required only one step. No chemiluminescence was observed from the bromo derivatives, and the detection limits of fluoropyrimidine compounds derivatized with the iodo compound and detected with peroxyoxalate chemiluminescence were in the low femtomole range.

Selective Transport of Histamine from a Mixture of Histidine and Histamine by the Use of Organic Liquid Membrane Systems

Abstract The transport behaviors of histidine (His) and its related compounds through organic liquid membranes were examined. The organic liquid membranes system was composed of two aqueous phases (Phases I and II) which were put on both sides of an organic layer containing a carrier. Chloroform and sodium di-2-ethylhcxyl sulfosuccinate (AOT) were used as the organic layer and the carrier, respectively. No transport reaction occurred without the carrier. The amounts of removal into the organic layer increased with an increase in the concentration of AOT up to 5 mM and maintained at higher concentrations. His and carnocine, which possess the carboxyl group, could be removed into the organic layer from Phase I at pH 4–5 but could not be removed at pH values higher than 5. On the other hand, histamine (Hm) and histidinol, which lack the carboxyl group, could be removed into the organic layer from Phase I at pH 7. Also, the compounds in the organic layer could be removed into Phase II at pH 10. On the basis o...

Precolumn derivatization for the determination of fluoropyrimidines by liquid chromatography with chemiluminescence detection

Abstract A method for the chemiluminescent detection of fluoropyrimidine compounds with 7-(diethylamino)-3- {4[(iodoacetyl)amino]phenyl}-4-methylcoumarin using peroxyoxalate is described. The procedure is rapid, simple and requires little or no experience in labelling techniques. The amounts of the derivatives are linearly related to the amount of the starting fluoropyrimidine compounds and the procedure can therefore be used in the determination of these solutes. Using reversed-phase liquid chromatography, detection levels of 20–40 fmol could be realized.

A Selective Determination of Hypoxanthine, Xanthine and Inosine by Reversed-Phase Liquid Chromatography Coupled with Enzyme Reactors

Abstract A Selective and sensitive assay of hypoxyanthine, xanthine and inosine by reversed-phase liquid chromatography coupled with immobilized enzyme reactors is described. The flourometric detection of hydrogen peroxide using immobilized peroxidase and p-hydroxyphenylacetic acid was applied to the assay of hypoxanthine, xanthine and inosine, which were oxidized to hydrogen peroxide in the presence of the immobilized enzymes (purine nucleoside phosphorylase and/or xanthine oxidase. The enzymes were immobilized the the intermolecular cross-linking method on controlled pore-glass. The method established was applied to serum and urine samples. The detection limits of hypoxanthine, xanthine and inosine were approximately 130, 300 and 650 pg per injection, respectively.

The use of xanthine oxidase for a specific and sensitive fluorimetric determination of 6-mercaptopurine in serum

A specific and highly sensitive fluorimetric method was developed for the determination of 6-mercaptopurine (6MP) in serum. The method is based on the enzymatic oxidation of 6MP with xanthine oxidase to the oxypurine, followed by oxidation with acidic chromate to the corresponding 6-sulfonate. The fluorescent product has excitation and emission maxima at 330 and 400 nm, respectively. The limit of sensitivity was approximately 22 pg/ml for 6MP in water. The sensitivity limit for 6MP in serum containing azathioprine was approximately 2.2 ng/ml. The rate constants for conversion of 6MP into the final product (6-thiouric acid) and the apparent Michaelis constant were also determined by a nonlinear regression analysis based on the integrated Michaelis-Menten equation using the ultraviolet absorbance data and the simplified complementary tristimulus colorimetry.

An Organic Liquid Membrane System for the Separation and Production of Histamine

Abstract The recovery of histamine (Hm) produced by histidine decarboxylase (HD) from histidine (His) by using an organic liquid membrane system was investigated. The system was composed of two aqueous phases (phase I of pH 4.5 and phase II of pH 7.2) separated by a third organic chloroform phase containing di-(2-ethylhexyl) phosphoric acid (EHP) as a carrier. His could not be moved from phase I of pH 4.5, the optimum pH of HD into the organic layer. On the other hand, Hm was moved from phase I into the organic layer, and Hm was released from the organic layer into phase II. The removal of Hm from phase I into the organic layer increased at dose dependency with EHP concentration up to 50 mM and decreased at concentrations above 50 mM. According to these results, the recovery of Hm from His by HD was investigated. In this experiment phase I was pH 4.5 containing 1 mM His, 40 μg/mL pyridoxal-5-phosphate, and a suitable concentration of HD; and phase II was set at pH 7.2. 170 μM Hm (3 h later) and 760 μM (8 ...

Evaluation of complementary color points in the identification of drugs by photodiode array detection in high-performance liquid chromatography

Abstract A system is described that identifies a number of unknown drugs in blood specimens for clinical-toxicological purposes. Reversed-phase (C 18 ) high-performance liquid chromatography with a photodiode array detector saves the ultraviolet (UV) spectrum of every chromatographically significant peak. The relative retention times could be used as one element of the identification; the second element is the UV spectrum, which is recorded on-line during the separation. The development of a computer program enabled complementary color points to be calculated from the UV spectrum. An evaluation of these color points for identifying the drugs has been made. Precisions of approximately 2% relative standard deviation can be achieved for complementary color points obtained from 18 different drugs. The combination of Chromatographic and complementary color points selects the most suitable drug, which could be the unknown.