Masaaki Kai

Fluorogenic reactions for biomedical chromatography

A number of fluorogenic reactions, which have been used for HPLC detection systems by means of pre- and/or postcolumn derivatization, are surveyed with respect to both sensitivity and selectivity for the determination of biomedically important substances. For the derivatization of the substances, two types of fluorogenic reactions, fluorescence-generating and fluorescence-tagging, have been studied. The former are usable in most instances for both pre- and postcolumn derivatization methods, and the latter only for precolumn derivatization methods. HPLC methods utilizing the fluorogenic reactions allow analytes to be detected at picomole-subfemtomole levels. In the fluorescence-generating reactions, several fluorogenic reagents possessing two or more reactive sites in the molecule, which show molecular recognition for a variety of analytes, permit facile and reproducible detection in HPLC because there are fewer interferences from biological matrices.

High-performance liquid chromatography of guanidino compounds using benzoin as a pre-column fluorescent derivatization reagent

Abstract A high-performance liquid chromatographic method is described for the simultaneous separation of nine guanidino compounds of biological importance. Guanidino compounds are converted into the corresponding fluorescent derivatives by reaction with benzoin, and separated within 25 min on a reversed-phase column, μBondapak Phenyl, with linear gradient elution using aqueous methanol containing a Tris—hydrochloric acid buffer (pH 8.5). The method is simple, rapid and sensitive; the lower limits of detection for the guanidino compounds are 20–100 fmol in a 100-μl injection volume.

High-performance liquid chromatography of tyrosine-containing peptides by pre-column derivatization involving formylation followed by fluorescence reaction with 1,2-diamino-4,5-dimethoxybenzene

A pre-column fluorescence derivatization method is described for the high-performance liquid chromatographic determination of tyrosine-containing peptides. A tyrosyl residue in the peptide is first formylated in an alkaline medium in the presence of chloroform, and the resulting aldehyde is then converted into a fluorescent derivative by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative is separated on a reversed-phase column (LiChrosorb RP-18) by isocratic elution with an aqueous acetonitrile-containing potassium chloride-hydrochloric acid buffer (pH 2.2) and sodium 1-hexanesulphonate. The method is selective and fairly sensitive; the lower limits of detection for the tyrosine-containing peptides tested are in the range 3.4-26.2 pmol in a 100-microliter injection volume.

On-line post-column fluorescence derivatization of arginine-containing peptides in high-performance 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 fluorogenic reagent for guanidino compounds, after separation on a reversed-phase column (TSKgel 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 fluorescence detection only shows peaks corresponding to arginine-containing peptides. The facile detection of arginine-containing fragments in the tryptic digest of beta-melanocyte stimulating hormone as a model compound could be achieved by comparison with a chromatogram obtained with ultraviolet absorption detection at 215 nm.

High-performance liquid chromatographic measurement of guanidino compounds of clinical importance in human urine and serum by pre-column fluorescence derivatization using benzoin

High-performance liquid chromatographic microanalyses for guanidino compounds in human physiological fluids have been accomplished by means of a pre-column fluorescence derivatization method using benzoin. The guanidino compounds in urine or deproteinized serum after ultrafiltration are converted to the fluorescent derivatives with benzoin in an alkaline medium, and the derivatives are separated simultaneously within 25 min on a reversed-phase column (mu Bondapak Phenyl) with a linear gradient elution of methanol in aqueous mobile phase (pH 8.5). The method permits the quantitative determination of guanidinosuccinic acid, methylguanidine, taurocyamine and guanidinobutyric acid at concentrations of as low as 8-78 pmol/ml in human urine and serum.

High-performance liquid chromatographic determination of free and total polyamines in human serum as fluorescamine derivatives

A highly sensitive and simple fluorimetric method for the determination of free and total polyamines, spermidine, spermine, putrescine and cadaverine, in human serum by high-performance liquid chromatography is described. The polyamines, obtained after clean-up of deproteinized serum by Cellex P column chromatography, are converted to their fluorescamine derivatives in the presence of nickel ion which inhibits the reaction of interfering amines with fluorescamine, and the derivatives are separated simultaneously by reversed-phase chromatography (LiChrosorb RP-18) with a linear gradient elution. The lower limits of detection are 10 and 15 pmole for spermine and the others in 0.5 ml of serum, respectively.

Selective determination of N-terminal tyrosine containing peptides by a novel fluorescence reaction with borate, hydroxylamine and cobalt(II)

Abstract A fluorimetric method is proposed for determining N -terminal tyrosine-containing peptides, of which some peptides such as enkephalins and kyotorphin are of physiological importance. An intense fluorescence is produced when the peptide is heated at 100°C for 3 min in a weakly alkaline medium containing borate, hydroxylamine and cobalt(II). The fluorescent species is stabilized with β-mercaptoethanol, with excitation and emission maxima at 335 and 430 nm, respectively. The method is highly selective for N -terminal tyrosine-containing peptides, with a detection limit of 43–69 pmol ml −1 .

High-performance liquid chromatographic analyser for guanidino compounds using benzoin as a fluorogenic reagent.

An automatic analyser based on high-performance liquid chromatography has been developed for the quantification of biogenic guanidino compounds in human physiological fluids. Fourteen guanidino compounds are mutually separated within 35 min on a cation-exchange column with a stepwise gradient elution of pH and/or ionic strength in the mobile phase and then converted automatically to their fluorescent derivatives with benzoin. The method in this system is simple, rapid and sensitive; the lower limits of detection are 5-50 pmol for monosubstituted guanidino compounds, 1 nmol for creatine and 20 nmol for creatinine in 100 microliter of injection volume.

Chemiluminescence determination of guanine and its nucleosides and nucleotides using phenylglyoxal

Abstract A novel chemiluminescence method is described for the determination of guanine and its nucleosides and nucleotides. The method is based on the fluorescence reaction of the substances with phenylglyoxal in a phosphate buffer (pH 6.0) at 37°C for 20 min, followed by a chemiluminescence reaction with N,N -dimethylformamide in a weakly alkaline medium. The established conditions of the chemiluminescence reaction do not permit any development of chemiluminescence from other nucleic acid bases such as adenine, cytosine, uracil and thymine, and their nucleos(t)ides. This method is quite selective and approximately 20-fold more sensitive than the fluorimetric one; the detection limits for guanine nucleos(t)ides are 4–19 pmol ml −1 in the reaction mixture.

High-performance liquid chromatography of N-terminal tryptophan-containing peptides with precolumn fluorescence derivatization with glyoxal

A precolumn fluorescence derivatization method combined with high-performance liquid chromatography is described for the sensitive and selective determination of N-terminal tryptophan-containing peptides. The peptides and tryptophan were converted into fluorescent derivatives with glyoxal in a moderately acidic medium (pH 4.5). The derivatives were separated on a reversed-phase column with isocratic elution with an aqueous mobile phase composed of acetonitrile, methanol and phosphate buffer (pH 6.0), and subsequently detected by fluorimetry. The derivatization technique provided the respective N-terminal tryptophan-containing oligopeptides with single fluorescent peaks in chromatography. The detection limits for the peptides were 55-382 fmol per 100-microliters injection volume at a signal-to-noise ratio of 3. The method also allowed the facile detection of an N-terminal tryptophyl fragment in the enzyme reaction mixture of dynorphin A with trypsin.