On-line Post-column Fluorescence Research Articles

Determination of guanine and its nucleosides and nucleotides in human erythrocytes by high-performance liquid chromatography with postcolumn fluorescence derivatization using phenylglyoxal reagent

A high-performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for the simple and sensitive determination of guanine and its nucleosides and nucleotides in human erythrocytes. After deproteinization of the biospecimen, guanine and its nucleosides and nucleotides were separated on a reversed-phase column (TSKgel ODS-120T) by gradient elution with methanol in the aqueous mobile phase consisting of tetra- n-propylammonium phosphate (pH 6.0) and phosphate buffer (pH 6.0). The compounds were then automatically converted into fluorescent derivatives by reaction with phenylglyoxal. This derivatization was selective for guanine-containing compounds. The present method permitted the reliable quantification of GDP and GTP in human erythrocytes. The detection limits (at a signal-to-noise ratio of 3) for guanine and its nucleosides and nucleotides were 3.2–10.0 pmol in a 20-μl injection volume. The concentrations of GDP and GTP in human erythrocytes were 17.2 ± 6.2 and 40.2 ± 5.8 nmol/ml, respectively.

Determination of Seven Opioid Peptides in Rat Brain by High Performance Liquid Chromatography with On-Line Postcolumn Fluorescence Derivatization

A high performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for a simple and sensitive determination of opioid peptides, including enkephalins in rat brain. The peptides were separated on a reversed-phase column (Asahipak ODP-50) by gradient elution of acetonitrile in the mobile phase containing a borate buffer (pH 10.0). They were then automatically converted into fluorescent derivatives by reactions with hydroxylamine, cobalt(II) ion and borate. This derivatization was specific for N-terminal tyrosine-containing peptides. It was successfully used not only for the simultaneous quantification of the endogenous seven opioid peptides at the picomole level in brain tissue, but also for their identification by means of enzymatic degradation using carboxypeptidase A and trypsin. The detection limits (S/N=3) for the synthetic peptides were 0.5–1.5 pmol in the injection volume.

High Performance Liquid Chromatographic Determination of Ribonucleotides by Postcolumn Derivatization Involving Oxidation Followed by Fluorescence Reaction, and Its Application to Human Erythrocyte Sample

A selective method for the determination of ribonucleotides is described which employs high performance liquid chromatography with on-line postcolumn fluorescence derivatization. Ribonucleotides are separated on a reversed-phased column (TSK gel ODS-80™) and automatically converted into fluorescent derivatives by reaction with 1,2-bis(4-methoxyphenyl)ethylenediamine in an acidic medium after periodate oxidation. The detection limits (S/N=3) for ribonucleotides are 14–67 pmol in a 100 μl injection volume. The quantification of ribonucleotides in human erythrocytes is also described.

Determination of Reducing Sugars by High Performance Liquid Chromatography with Postcolumn Fluorescence Derivatization Using 1,2-Bis(4-methoxyphenyl)ethylenediamine

A sensitive method for the determination of reducing sugars is described which employs high performance liquid chromatography with on-line postcolumn fluorescence derivatization. Reducing sugars are separated by borate complex anion-exchange chromatography on a trimethylammonium-bonded resin column (TSK gel Sugar AXG) and automatically converted into fluorescent derivatives by reaction with 1,2-bis(4-methoxyphenyl)ethylenediamine in an alkaline medium. The detection limits (S//V=3) for reducing sugars are 30- 160 pmol in a 100-μl injection volume. The quantification of reducing sugars in human urine and serum is also described.

High-performance liquid chromatographic determination of substance p-like arginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of substance P, one of the neuropeptides, in the hypothalamus tissue of rat brain. The detection is based on on-line post-column fluorescence derivatization selective for arginine-containing peptides. The endogenous substance P-like arginine-containing peptide extracted from the tissue and [D-Phe11]-neurotensin as an internal standard were separated from various interfering substances on a reversed-phase column (TSKgel ODS-120T) by gradient elution with acetonitrile-phosphate buffer (pH 2.3). The peptides in the eluate were then automatically converted into fluorescent derivatives for detection by reaction with benzoin. Arginine-containing fragments produced by the enzyme reaction of substance P in the chromatographic fraction with trypsin were also detected, for the identification of the endogenous substance P-like arginine-containing peptide. The method was sensitive enough to permit the quantitative determination of the peptide at a concentration as low as 580 fmol/mg of protein in the brain homogenate. The concentration values of the substance P-like arginine-containing peptide in the tissue were 9.45 +/- 1.50 pmol/mg of protein (six determinations).

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephalin, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5–20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54–191 pmol/min·mg protein for enkephalinase A and 79–153 pmol/min·mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.

On-line post-column fluorescence derivatization of arginine-containing peptides in high-perfomance liquid chromatography

A selective detection system based on on-line post-column fluorescence derivatization is described for the analysis of arginine-containing peptides by high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives with benzoin, a fluoregenic reagent for guarnidino compounds, after separation on a reversed-phase column (TSK gel ODS-120T) and detection in an ultraviolet absorption detector. The system permits fluorescence detection at 435 nm emission with irradiation at 325 nm for arginine-containing peptides in as little as picomole amounts. The chromatogram obtained with fluroscence detection only shows peaks corresponding to arginine-containing peptides. The facile detection of arginine-containing fragments in the tryptic digest of β-melanocyte stimulating hormone as a model compound could be achieved by comparison with a chromatogram obtained with ultraviolet absorption detection at 215 nm.