Tokishi Hayashi

Determination of free amino acid enantiomers in rat brain and serum by high performance liquid chromatography after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde

The concurrent determination of free amino acid enantiomers and non-chiral amino acids in rat brain and serum was accomplished by high-performance liquid chromatography with fluorimetric detection after derivatization with N-tert.-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. The method revealed the presence of a large amount of free D-serine (0.22 mumol/g of tissue; D/D + L ratio = 0.25) in the brain whereas D-aspartate and D-alanine were established to be at trace levels. These results further support the presence of D-serine in adult brain tissues as demonstrated by recent work using gas chromatography.

High-performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin as fluorescence reagent

Abstract A sytem for the high-performance liquid chromatography of carboxylic acids using 4-bromomethyl-7-acetoxycoumarin (Br-Mac) as the fluorescence reagents is described. Br-Mac reacts with carboxylic acids to give the ester derivatives, which are separated using reversed-phase liquid chromatography. The eluate from the column is mixed with an alkaline solution. The labelled carboxylic acids are hydrolysed to the fluorescent coumarin derivatives, which are introduced into a flow-through fluorimeter. In this system, the fluorescent hydrolysate, equimolar to a carboxylic acid, is detected and this fluorophor is common to every carboxylic acid. Only a slight variation is found in the peak areas for various carboxylic acids. A gradient elution technique is effectively used in this system because the fluorescence quantum yield of the fluorescent hydrolysate is not affected by the constitution of the mobile phase. Low femtomole levels of carboxylic acids can be detected.

High-performance liquid chromatographic determination of α-keto acids in human urine and plasma

A high-performance liquid chromatographic method has been developed for the determination of α-keto acids in human urine and plasma. These acids were prepurified using a column of hydrazide gel and derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into ethyl acetate. The 2-quinoxialinol derivatives were separated by reversed-phase paired-ion chromatography using a 250 × 4 mm-i.d. column packed with LiChrosorb RP-8 (5 μm). This method is sensitive, selective, and reproducible. The α-keto acids in urine and plasma from normal individuals were determined.

High-performance liquid chromatographic determination of pyruvic acid and α-ketoglutaric acid in serum

A high-performance liquid chromatographic determination of pyruvic acid and alpha-ketoglutaric acid in serum is described which is based on the formation of 2,4-dinitrophenylhydrazones. The syn-anti isomerization of the 2,4-dinitrophenylhydrazone of pyruvic acid is also discussed.

Simultaneous separation and sensitive determination of free fatty acids in blood plasma by high-performance liquid chromatography

Fatty acids are separated by reversed-phase high-performance liquid chromatography after derivatization with a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin. Each derivative eluted from a column is successively hydrolysed by mixing it with an alkaline solution, and the produced fluorescence is detected. The derivatives of series of both saturated and unsaturated fatty acids (C6:0--C20:4) are simultaneously separated by a continuous gradient elution method using a methanol-based solvent containing acetonitrile. The quantitative detection of fatty acids is over a range of 5-1000 pmol per derivatization mixture. This method is applicable to the quantitative analysis of free fatty acids in normal human blood samples and blood samples from diabetic patients. Ten microliters of blood plasma are sufficient to carry out the determination. The analytical results show good recovery and good reproducibility. This sensitive method is very useful for the analysis of fatty acids in very low concentrations.

Sensitive high-performance liquid chromatographic method for prostaglandins using a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin

High-performance liquid chromatography of prostaglandins is developed in which a fluorescence reagent, 4-bromomethyl-7-acetoxycoumarin is used to perform the high-sensitivity detection. The reagent reacts with prostaglandins and related compounds to form the ester derivatives, which are separated using a reversed-phase system. Each labeled compound eluted from the column is successively hydrolyzed to the fluorescent coumarin derivative, and this fluorophore is introduced into a flow-through fluorometer. Prostaglandins can be determined in the range of at least 1 nmol to 5 pmol, and the detection limit is about 10 fmol. This system is applied to the analysis of prostaglandins in human seminal fluid.

High-performance liquid chromatographic determination of α-keto acids in plasma with fluorometric detection

This paper describes a sensitive high-performance liquid chromatographic method for the quantitative determination of alpha-keto acids in plasma using a fluorescence detector. This method is about ten times more sensitive than that reported in a previous paper. Only 50 microliters of plasma are needed for the determination of alpha-keto acids. However, p-hydroxyphenylpyruvic acid could not be analysed because the quinoxalinol derived from it does not exhibit fluorescence.

High-performance liquid chromatographic determination of urinary catecholamines by pre-column solid-phase dansylation on alumina

Sensitive and selective high-performance liquid chromatographic determination of catecholamines by pre-column solid-phase dansylation is described. After catecholamines are adsorbed on alumina, the amino groups not responsible for adsorption are dansylated by a solid-phase reaction. The excess reagent and fluorescent contaminants are washed out, and the dansylated catecholamines are eluted and separated by reversed-phase high-performance liquid chromatography. The four catecholamine derivatives can be separated within 10 min and no major interfering peak is observed on chromatograms. The response of each catecholamine is linear from 10 to 500 pmol per sample and the detection limit is 0.5 pmol. This method was applied to determination of catecholamines in human urine.

High-speed liquid chromatographic determination of phenylpyruvic acid

A high-speed liquid chromatographic method has been developed for the determination of urinary phenylpyruvic acid. This acid is converted by treatment with naphthalene-2,3-diamine into 3-benzyl-2-hydroxybenzoquinoxaline, which is extracted into carbon tetrachloride for separation. 2-Mercaptoethanol is a useful stabilizer, and 2-chlorothioxanthone is a suitable internal standard. The method is specific, and the results are not affected by the presence of such 2-oxo-acids as pyruvic acid, 2-oxobutyric acid, 2-oxoglutaric acid, and 4-hydroxphenylpyruvic acid.

Determination of tetrahydro-β-carbolines in urine by high-performance liquid chromatography with suppression of artefact formation

A high-performance liquid chromatographic method has been developed for the determination of urinary tetrahydro-beta-carbolines. When standing tryptamine with formaldehyde and acetaldehyde under extraction conditions, the significant amounts of artefact 1,2,3,4-tetrahydro-beta-carboline (TBC) and 1-methyl-1,2,3,4-tetrahydro-beta-carboline (MTBC) were formed in a short time. Urine samples added with 2-ethyl-1,2,3,4-tetrahydro-beta-carboline (an internal standard) were treated with fluorescamine, and then with glycine, followed by serial solvent extractions. Such a pretreatment using two-step reactions removed a precursor (trypamine) by extracting its fluorescamine derivative, and enhanced the detection response by consuming excess fluorescamine. It solved the analytical problem that artefact TBC and MTBC are formed during analysis. Reversed-phase ion-pair chromatography using a C8-column and trifluoroacetic acid as a counter ion completed a base-line separation of three analytes within 10 min. The calibration graphs showed a good linearity in the range 0.1-50.0 ng ml-1 of urine samples spiked with standard TBC and MTBC. In the spike experiment, the recovery and relative standard deviation were almost 100% and less than 3.0%, respectively, for both TBC and MTBC. The proposed method enables the determination of the genuine urinary concentrations of TBC and MTBC without involving their artefacts.