Shingo Hirose

Use of 4-bromomethyl-7-methoxycoumarin for derivatization of pyrimidine compounds in serum analysed by high-performance liquid chromatography with fluorimetric detection

Derivatization of the pyrimidine nucleobases and nucleosides with 4-bromomethyl-7-methoxycoumarin was studied with the aim of developing a sensitive and selective column liquid chromatographic method for these substances in serum. The labeling reactions and the nature of derivatives are discussed, together with the chromatographic properties of these derivatives. The derivatives are stable for at least several weeks. Typical detection limits are 50 pg for inosine, 150 pg for uridine, 50 pg for uracil, 50 pg for thymine and 100 pg for fluorodeoxyuridine, respectively. Within-day coefficients of variation averaged 5.0% for the stored-frozen serum pools; the mean day-to-day value was 5.2%. Thirty samples could be processed per working day.

Simultaneous assay of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma using high-performance liquid chromatography

We devised a procedure for the simultaneous determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites using a reversed-phase liquid chromatographic system. Detection is achieved by an electrochemical detector and a fluorescence detector connected in series. Sample preparation is kept to a minimum, and involves precipitation of proteins with trichloroacetic acid and perchloric acid, and subsequent neutralization, thus omitting the commonly adopted adsorption step. Chromatographic peaks were identified on the basis of retention behaviour and the ratio of responses at several oxidation potentials. The method was applied to the quantitative determination of 3,4-dihydroxyphenylalanine, catecholamines and O-methylated metabolites in human plasma.

Comparison of age-associated changes of c-myc gene methylation in liver between man and mouse

Age-associated changes in DNA methylation of the c-myc gene in liver were compared between humans and mice which have large differences in aging rate. Although the overall methylation profiles of the gene and their age-related changes were found to be similar, the rate of change was much slower in humans than in mice. It suggests that the age-associated alteration of c-myc gene methylation is closely related to biological aging rather than to chronological aging.

Determination of the aminoglycoside antibiotics sisomicin and netilmicin in dried blood spots on filter discs, by high-performance liquid chromatography with pre-column derivatization and fluorimetric detection

A simple method for the determination of the aminoglycoside antibiotic sisomicin or its 1-N-ethyl derivative, netilmicin in whole blood, using dried blood spots (DBSs) on filter-paper punched discs has been developed. Sisomicin or netilmicin in the DBSs were recovered most effectively in 0.5 M Na2HPO4 using ultrasonication. The eluates from the DBSs were treated by ultrafiltration for deproteinization and subjected to pre-column fluorescent derivatization using o-phthalaldehyde and beta-mercaptopropionic acid in 0.05 M KH2PO4-borate buffer (pH 9.0), followed by determination by reversed-phase high-performance liquid chromatography. The detection limits of sisomicin and netilmicin in the DBSs on punched discs (10.1 microliters of whole blood) were 0.053 and 0.50 micrograms per ml of whole blood, respectively (signal-to-noise ratio greater than or equal to 2). The method permits a simple collection of blood at the microlitre level and should prove particularly useful for monitoring sisomicin and netilmicin in blood at therapeutic levels in geriatric and paediatric patients.

Determination of total 3α-hydroxy bile acids in serum by a bioluminescent flow-injection system using a hollow-fibre reactor

Abstract A continuous-flow bioluminescence method for measuring total 3α-hydroxy bile acids in serum is described. A bacterial luciferase and NADH:FMN oxidoreductase are covalently co-immobilized on a CNBr-activated Sepharose 4B. A permeable membrane (hollow fibre) reactor is used for the introduction of NAD + and bioluminescent reagent. The reagent permeates into the flow-stream due to the existing difference in ionic strength. The continuous-flow light-emitting system, in which the column filled with the immobilized bioluminescent enzyme is placed in front of a photomultiplier tube inside a photon counter, is versatile and simple. The technique was tested by comparing results with those obtained by fluorimetry. More than 20 samples an hour can be analyzed. Normal values for total bile acids content serum ranged from 1.0 to 7.5 μM, in agreement with those obtained by the other method. Excellent reproducibility, precision, and sensitivity are achieved.

Determination of m- and p-O-methylated products of l-3,4-dihydroxyphenylalanine using high-performance liquid chromatography and electrochemical detection

In a study of in vitro and in vivo metabolism of L-3,4-dihydroxyphenylalanine (L-Dopa), two methods of high-performance liquid chromatography (HPLC) were used to separate the m- and p-O-methylated products. A reversed-phase column and an aqueous mobile phase by gradient elution were used; the elute was analyzed electrochemically with a single amperometric and dual coulometric electrode. The L-Dopa and its O-methylated products could be detected individually in the enzymatic methylation of rat liver homogenate and in patients with Parkinsons disease. Meta/para ratios of O-methylation are easily obtained by this method.

Fluorescence determination of 5-fluorouracil and 1-(tetrahydro-2-furanyl)-5-fluorouracil in blood serum by high-performance liquid chromatography

Fluorescence derivatization of 5-fluorouracil (5-FU) and 1-(tetrahydro-2-furanyl)-5-fluorouracil (ftorafur, FT) with 4-bromomethyl-7-methoxycoumarin using 18-crown-6 as a catalyst is studied with aim of developing a sensitive and selective liquid chromatographic method. 5-FU and FT form virtually substituted derivatives which possess maxima in their fluorescence emission spectra near 400 nm. These derivatives are separated by thin-layer chromatography and high-performance liquid chromatography to confirm the completion of reaction. For the determination of 5-FU and FT in serum, the reversed-phase high-performance liquid chromatographic separation of the derivatives is studied with a C13 column. This chromatography is of importance for the accurate determination of 5-FU and FT, which are, respectively, an important antitumour agent for the treatment of solid tumours in clinical medicine and a masked form of 5-FU generated in vivo.

Rapid and sensitive on-line precolumn purification and high-performance liquid chromatographic assay for bile acids in serum

A simple and rapid technique for the simultaneous isolation and analysis of fifteen kinds of bile acid was developed using reversed-phase high-performance liquid chromatography with an automatic dual-precolumn switching system. The serum samples were directly injected onto a first precolumn (hydroxyapatite), which was flushed with 1 mM phosphate buffer. Serum proteins were strongly retained on the hydroxyapatite column, but bile acids were unretained. The bile acids were absorbed on a second precolumn (Serumout-25) and eluted onto the analytical column with solvent B (acetonitrile-methanol-30 mM ammonium acetate, 20:20:60, v/v/v). For the separation of each bile acid, the gradient elution technique was used (solvent A was acetonitrile-methanol-30 mM ammonium acetate, 30:30:40). After separation of the bile acids, NADH was produced by use of immobilized 3 alpha-hydroxysteroid dehydrogenase column and then determined fluorimetrically (gamma em = 460 nm, gamma ex = 350 nm). The recoveries of bile acids in serum generally approached 100%.

Pre-column derivatization of sisomicin with o-phthalaldehyde-β-mercaptopropionic acid and its application to sensitive high-performance liquid chromatographic determination with fluorimetric detection

The stability of the o-phthalaldehyde (OPA) derivatives of sisomicin obtained using beta-mercaptopropionic acid was investigated by reversed-phase high-performance liquid chromatography. One of the fluorescent derivatives of sisomicin was stable at least for 6 h in 50% methanol under the optimal conditions used (OPA concentration, pH and temperature). When plasma samples spiked with sisomicin were analysed, the response was linear in the calibration range 136-900 pg of sisomicin per injected volume (40 microliters). As little as 0.06 micrograms of sisomicin per 1 ml of plasma could be detected with signal-to-noise ratio greater than or equal to 2. For plasma samples spiked with 0.2 micrograms/ml sisomicin, the recovery was 97.1 +/- 6.6% (mean +/- S.D., n = 5) with a within-run coefficient of variation of 6.8% and a day-to-day coefficient of variation of 7.2%. The method was also applied to plasma samples from rabbit after a subcutaneous injection of 1 mg/kg sisomicin.

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.