Yasuto Tsuruta

Sensitive derivatization reagents for hydroxyl and amino compounds for thin-layer or high-performance liquid chromatography with fluorescence detection

Abstract Three fluorescent derivatization reagents for compounds having hydroxyl and/or amino groups are described. 4-(2-Phthalimidyl)benzoyl chloride, 3-(2-phthalimidyl)benzoyl chloride and 3-(2-phthalimidyl)-4-methoxybenzoyl chloride, prepared from the corresponding phthalimidylbenzoic acid, were stable at room temperature and condensed quantitatively with alcohols, amines and amino acids in the presence of alkali under mild conditions to give strongly fluorescent derivatives. The derivatives were separated by thin-layer and high-performance liquid chromatography.

Determination of nicotinic acid in serum by high-performance liquid chromatography with fluorescence detection.

A sensitive method for the determination of nicotinic acid in serum is described which employs high-performance liquid chromatography with fluorescence detection. Nicotinic acid and 2-chloronicotinic acid as an internal standard in deproteinized serum are reacted with N,N-dicyclohexyl-O-(7-methoxycoumarin-4-yl)methylisourea in acetone to give the corresponding fluorescent 4-hydroxymethyl-7-methoxycoumarin esters. The compounds are separated by reversed-phase chromatography on LiChrosorb RP-18 with isocratic elution using aqueous acetonitrile containing a small amount of sodium 1-hexanesulphonate as a mobile phase. The detection limit of nicotinic acid in serum was 0.2 nmol/ml. The method requires only 100 microliters of serum.

Determination of taurine in plasma by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A sensitive high-performance liquid chromatography method for the determination of taurine in human plasma was developed. Taurine and N-methyltaurine (internal standard) were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride to produce fluorescent sulfonamides. The labeling reaction was carried out at 70 degrees C for 20 min at pH 7.5. The fluorescent derivatives were separated on a reversed-phase column by a stepwise elution using (A) acidic phosphate buffer/acetonitrile (83/17) and (B) acetonitrile and detected by fluorescence measurement at excitation and emission wavelengths of 318 and 392 nm, respectively. The detection limit (signal-to-noise ratio=3) of taurine was 3 fmol per injection. The within-day and day-to-day relative standard deviations were 3.0-4.8 and 2.5-4.7%, respectively. The concentration (means) of taurine in normal human plasma was 48.9+/-7.5 microM.

Highly sensitive determination of N-terminal prolyl dipeptides, proline and hydroxyproline in urine by high-performance liquid chromatography using a new fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive pre-column HPLC method for simultaneous determination of prolyl dipeptides, Pro and Hyp in urine was developed. The analytes were labelled with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 20 min. The derivatives separated on tandem reversed-phase columns by a gradient elution and were monitored with fluorescence detection at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides, Pro and Hyp were 1-5 fmol/injection (S/N = 3). Urine samples were treated with o-phthalaldehyde, followed by purification on a Bond Elut C18 column before conducting the labelling reaction. Pro-Hyp, Pro-Gly and Pro-Pro were identified as prolyl dipeptides in urine. The within-day and between-day relative standard deviations were 1.5-4.8 and 1.7-5.8%, respectively. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human urine were 97.6 +/- 28.2, 2.74 +/- 1.48, 2.08 +/- 1.13, 6.71 +/- 3.34 and 2.30 +/- 1.59 nmol/mg creatinine, respectively.

Fluorometric determination of N-terminal prolyl dipeptides, proline and hydroxyproline in human serum by pre-column high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride

A highly sensitive HPLC method for the determination of prolyl dipeptides, Pro and Hyp in serum was developed. After deproteinization of serum and pretreatment with o-phthalaldehyde, the analytes were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 10 min. The fluorescent derivatives of prolyl dipeptides, Pro and Hyp, were separated on tandem reversed-phase columns by a gradient elution at 55 degrees C and detected by fluorescence measured at 318 nm (excitation) and 392 nm (emission). The detection limits for prolyl dipeptides were 2-5 fmol/injection (S/N=3). Pro-Hyp, Pro-Gly and Pro-Pro were identified as serum prolyl dipeptides. The within-day and between-day relative standard deviations were 1.5-7.9 and 2.4-10.8%, respectively. The recoveries were in the range of 90.8-97.3%. The concentrations of Pro-Hyp, Pro-Gly, Pro-Pro, Pro and Hyp in normal human serum (n = 10) were 0.64+/-0.35, 0.078+/-0.047, 0.022+/-0.016, 177.0+/-43.0 and 11.1+/-3.5 microM, respectively. The concentrations of Pro-Hyp and Pro-Pro in serum of a patient with bone metastases of prostatic cancer were about three times and 50 times, respectively, higher than those in normal human serum.

Phthalimidylbenzenesulphonyl chlorides as fluorescence labelling reagents for amino acids in high-performance liquid chromatography

This paper deals with the preparations of 4-(N-phthalimidyl) benzenesulphonyl chloride and 2-methoxy-5(N-phthalimidyl) benzenesulphonyl chloride as fluorescent derivatization reagents for amines and amino acids and their reactivities towards amino compounds using thin-layer chromatography (TLC) and HPLC

Simultaneous determination of serum and urinary hydroxyproline and proline by liquid chromatography using two fluorescent labeling reagents

A method for the simultaneous determination of free serum and total urinary hydroxyproline (Hyp) and proline (Pro) concentrations by liquid chromatography (LC) using two fluorescent labeling reagents was developed. Serum Hyp and Pro, after treatment with o-phthalaldehyde, were derivatized with 4-(2-phthalimidinyl)phenylsulfonyl chloride (Phisyl-Cl). Imino acids in hydrolysed urine were derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl chloride (DPS-Cl) after treatment with o-phthalaldehyde and cleanup on a Bond Elut C 18 column. The two reaction mixtures were combined and mixed with dichloromethane to extract the excess of the reagents, and then the aqueous layer (10 pl) was subjected to LC. The Phisyl and DPS derivatives of imino acids were separated on a reversed-phase column by gradient elution with phosphate buffer (5 mM, pH 2.5) and acetonitrile at 35°C and detected by fluorescence measurement at 300 nm (excitation) and 400 nm (emission). The detection limits (s:n=3) for Hyp and Pro were 30 and 40 fmol/injection, respectively, for Phisyl derivatives and 5 fmol/injection for both DPS derivatives. The within-day and day-to-day relative standard deviations for Hyp and Pro in serum and urine were <2.8%. The recoveries of Hyp and Pro added to serum and urine were 97.6-103.6%.

Sensitive determination of carnosine in urine by high-performance liquid chromatography using 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride as a fluorescent labeling reagent

A simple and highly sensitive high-performance liquid chromatography procedure was developed for the determination of carnosine in urine. Carnosine was derivatized with 4-(5,6-dimethoxy-2-phthalimidinyl)-2-methoxyphenylsulfonyl chloride at 70 degrees C for 15 min in borate buffer (20 mmol l(-1), pH 9.0) to produce fluorescent sulfonamides. After hydrolysis of the reaction mixture with formic acid at 100 degrees C for 15 min, the fluorescent derivative of carnosine was separated on a reversed-phase column with a linear gradient elution using solvents of (A) acetate buffer (0.1 mmol l(-1), pH 7.0) and (B) acetonitrile at a flow-rate of 1.0 ml/min and was detected at excitation and emission wavelengths of 318 and 400 nm, respectively. The detection limit of carnosine was 4 fmol at a signal-to-noise ratio of 3. The within-day and day-to-day relative standard deviations were 2.7-4.6% and 0.4-5.2%, respectively. The concentration of carnosine in normal human urine was found to be 4.6-125 nmol (mg creatinine)(-1) (mean+/-SD: 21.6+/-26.6 nmol (mg creatinine)(-1), n=20).

Fluorescence reaction of bilirubin with zinc ion in dimethyl sulfoxide and its application to assay of total bilirubin in serum

Abstract A new fluorimetric method is described for the determination of total bilirubin in serum. The fluorescence with maximum excitation and emission at 470 and 545xa0nm, respectively, is based on the reaction of bilirubin with zinc acetate in dimethyl sulfoxide (DMSO). Serum (10xa0μl) is added to 2.5xa0ml of DMSO solution containing zinc acetate (0.01xa0M) and Tris (0.2xa0M). After 60xa0min, at 37°C, the fluorescence is measured. The blank is obtained by mixing serum (10xa0μl) and Tris-DMSO solution (2.5xa0ml) without zinc acetate. The method gave a linear calibration for 0.1–5xa0mgxa0dl−1 bilirubin, a 3.3σ detection limit of 5xa0μgxa0dl−1 and relative standard deviations for bilirubin in control sera of 1–2% (n=10). It is simple, specific and sensitive, and showed good correlation with the usual diazo-coupling method.

4-(5,6-Dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide as a fluorescent labelling reagent for determination of carboxylic acids in precolumn high-performance liquid chromatography

A fluorescent labelling reagent, 4-(5,6-dimethoxy-2-phthalimidinyl)phenylsulfonyl semipiperazide, was designed for the determination of carboxylic acids by precolumn HPLC. The reagent was reacted with carboxylic acids at 100 degrees C for 15 min in the presence of an activating reagent (a mixture of triphenylphosphine, 2,2-dipyridyldisulfide and pyridine) and produced highly fluorescent derivatives, which were separated on a reversed-phase column by fluorescence measurement at 317 nm (excitation) and 380 nm (emission). The detection limits were 4-12 fmol/injection. The reagent was used for HPLC assays of long chain fatty acids in human serum by isocratic elution.