Shuuji Hara

Fluorometric high-performance liquid chromatography of N-acetyl- and N-glycolylneuraminic acids and its application to their microdetermination in human and animal sera, glycoproteins, and glycolipids

A simple, rapid, and highly sensitive high-performance liquid chromatographic method is described for the determination of N-acetyl- and N-glycolylneuraminic acids in human and animal sera, glycoproteins, and glycolipids. The neuraminic acids, released by acid hydrolysis of these biological samples, are converted in dilute sulfuric acid with 1,2-diamino-4,5-methylene-dioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated within 12 min on a reversed-phase column (Radial-Pak cartridge C18) with an isocratic elution and detected fluorometrically. The detection limits are 25 fmol (7.7 pg) for N-acetylneuraminic acid and 23 fmol (7.5 pg) for N-glycolylneuraminic acid in a 10-microliter injection volume at a signal-to-noise ratio of 2. This method permits precise determination of the neuraminic acids in 5 microliter of human and animal sera or in 0.25-2.5 micrograms of glycoproteins and glycolipids.

Determination of mono-O-acetylated N-acetylneuraminic acids in human and rat sera by fluorometric high-performance liquid chromatography

A simple, rapid, and highly sensitive fluorometric high-performance liquid chromatographic method for the determination of N-acetylneuraminic acid and its mono-O-acetyl derivatives in human and rat sera is described. The neuraminic acids, released by hydrolysis of serum in 2 M acetic acid, are converted with 1,2-diamino-4,5-methylenedioxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives without the occurrence of O-acetyl migration and de-O-acetylation. The derivatives are separated isocratically within 25 min by reversed-phase chromatography using a TSK gel ODS-120T column. The limits of detection are 57-192 fmol in a 10-microliters injection volume at a signal-to-noise ratio of 3. This sensitivity permits precise determination of the neuraminic acids in 5 microliters of human and rat sera.

Highly sensitive determination of N-acetyl- and N-glycolylneuraminic acids in human serum and urine and rat serum by reversed-phase liquid chromatography with fluorescence detection

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of N-acetyl- and N-glycolylneuraminic acids in serum and urine is described. The neuraminic acids, released by hydrolysis of serum and urine, are converted in dilute sulphuric acid with 1,2-diamino-4,5-dimethoxybenzene, a fluorogenic reagent for alpha-keto acids, to highly fluorescent derivatives. The derivatives are separated isocratically within 8 min by reversed-phase chromatography using a Radial-Pak cartridge C18 column and detected fluorimetrically. The limit of detection is 40 fmol (12 pg) for both neuraminic acids in 10-microliters injection volume [0.3 nmol (90 ng)/ml) of serum or urine]. This sensitivity permits the precise determination of the neuraminic acids in 5 microliters of serum or urine. The method was applied to the determination of the neuraminic acids in sera from normal subjects and cancer patients, normal urine and rat serum.

3-Bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone as a new fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography

Abstract 3-Bromomethyl-6,7-dimethoxy-1-methyl-2(1 H )-quinoxalinone was found to be a selective and highly sensitive fluorescence derivatization reagent for carboxylic acids in high-performance liquid chromatography. Its reactivity was investigated for various linear C 3 C 20 saturated fatty acids. The reagent reacts with the fatty acids in acetonitrile in the presence of 18-crown-6 and potassium carbonate to produce the corresponding fluorescent esters, which can be separated on a reversed-phase column, Radial-Pak C 18 cartridge, with gradient elution using 57–100% (v/v) aqueous methanol; the detection limits for the acids were 0.3–1 fmol for an injection volume of 5 μl. The reagent also reacts with unsaturated fatty, dicarboxylic, aromatic carboxylic and hydroxycarboxylic acids and acidic nucleotides to form fluorescent derivatives. α-Keto acids and α-amino acids do not give fluorescent derivatives under these conditions.

6,7-Dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide: a highly sensitive fluorescence derivatisation reagent for carboxylic acids in high-performance liquid chromatography

6,7-Dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionylcarboxylic acid hydrazide was found to be a highly sensitive fluorescence derivatisation reagent for carboxylic acids in high-performance liquid chromatography. The reaction conditions were optimised for various C5–C20 saturated fatty acids. The reagent readily reacted with the fatty acids in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at room temperature to produce the corresponding fluorescent derivatives, which were separated on a reversed-phase column, Biofine RPC-SC 18 B, by gradient elution with 40–100% aqueous methanol. The derivatives were detected spectrofluorimetrically at 447 nm with excitation at 365 nm. The detection limits (signal to noise ratio = 3) for the acids were 3–6 fmol for an injection volume of 10 µl. The reagent was also applied to the derivatisation of some metabolites of arachidonic acid.

Highly sensitive determination of free fatty acids in human serum by high-performance liquid chromatography with fluorescence detection

A highly sensitive and rapid high-performance liquid chromatographic method for the determination of free fatty acids in human serum is described. The fatty acids are converted into the corresponding fluorescent derivatives by the reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 44 min on a reversed-phase column (YMC-Pack C8) with a gradient elution of aqueous methanol and detected fluorimetrically. The detection limits are 0.5-2 fmol in a 10-microliters injection volume. This sensitivity permits precise determination of free fatty acids including lauric, myristoleic and linolenic acids, which occur in serum at very low concentrations, in 5 microliters of sera from healthy subjects and patients with diabetes.

1,2-diamino-4,5-methylenedioxybenzene as a highly sensitive fluorogenic reagent for α-dicarbonyl compounds

Abstract The reactivity of methylglyoxal with 1,2-diaminobenzene derivatives is investigated in detail, to find fluorogenic reagents for α-dicarbonyl compounds. Of the eight derivatives tested, 1,2- diamino-4,5-methylenedioxybenzene is shown to be the best reagent in terms of reactivity, selectivity and sensitivity. The reagent reacts with α-dicarbonyl compounds in very dilute hydrochloric acid and the products fluoresce intensely in neutral and alkaline media. The derivatives of seven α-dicarbonyl compounds are separated by reversed-phase h.p.l.c. The detection limits are in the range 65–280 fmol per 10-μl injection volume. The fluorescent products are characterized as the corresponding 2- and or 3-substituted 6,7-methylenedioxyquinoxalines.

Determination of α-keto acids in serum and urine by high-performance liquid chromatography with fluorescence detection

A simple, rapid and highly sensitive high-performance liquid chromatographic method for the determination of α-keto acids in serum and urine is described. In dilute hydrochloric acid, α-keto acids are converted by 1,2-diamino-4,5-dimethoxybenzene into highly fluorescent quinoxalinone derivatives. The derivatives are isocratically separated simultaneously within 14 min by reversed-phase chromatography on a Radial-Pak cartridge C18 and detected fluorimetrically. The limits of detection are 10–300 fmol in an injection volume of 10 μl (40–1200 pmol/ml of serum or urine). This sensitivity permits precise determination of several α-keto acids in 5 μl of serum or urine from healthy persons, and also the determination of phenylpyruvic acid in normal urine which cannot be simultaneously determined by other methods.

Simple and sensitive HPLC method for the fluorometric determination of methotrexate and its major metabolites in human plasma by post-column photochemical reaction.

A simple and sensitive HPLC method has been developed for the determination of methotrexate (MTX) and its major metabolites, 7-hydroxymethotrexate (7-OH-MTX) and 2,4-diamino-N(10-) methylpteroic acid (DAMPA), in human plasma. After deproteinization of the plasma with 5% aqueous acetonitrile solution containing 5% trichloroacetic acid, MTX, 7-OH-MTX, DAMPA and 2,4-diaminopteroic acid (DAPA) as an internal standard were separated on a reversed-phase column, and the eluent was subsequently irradiated with UV light (245 nm), producing fluorescent photolytic degradation products. The analytes were then detected spectrofluorometrically at 452 nm with excitation at 368 nm. The extraction efficiencies of MTX, 7-OH-MTX and DAMPA from plasma at 100 pmol/mL were 81.5±5.4, 82.5±5.3 and 56.2±7.0%, respectively. The limits of quantification for MTX, 7-OH-MTX and DAMPA in plasma were 5 pmol (2.3 ng), 0.8 pmol (0.38 ng) and 10 pmol (3.4 ng)/mL, respectively. The within- and between-day variations for MTX, 7-OH-MTX and DAMPA were reliable (each was lower than 6.3%). This method was also used to monitor the concentrations of MTX and its metabolites in a patient on MTX therapy.

Fluorimetric high-performance liquid chromatography of prostaglandins and its application to their determination in human seminal fluid

A highly sensitive and simple high-performance liquid chromatographic method with fluorescence detection for the determination of prostaglandins is described. Prostaglandins are converted into the corresponding fluorescent derivatives by reaction with 3-bromomethyl-6,7-dimethoxy-1-methyl-2(1H)-quinoxalinone in the presence of potassium hydrogen carbonate and 18-crown-6 in acetonitrile. The derivatives are separated simultaneously within 34 min (the total run time per injection, 56 min) on a reversed-phase column (YMC Pack C8) by a stepwise elution with mixtures of acetonitrile, methanol and water and detected fluorimetrically. The detection limits are 10-15 fmol at a signal-to-noise ratio of 5 in a 10-microliter injection volume. Prostaglandins E1, E2, F1 alpha and F2 alpha in human seminal fluids are measured by this method.