Ichiro Ono

Structure determination of metabolites isolated from urine and bile after administration of AY4166, a novel d-phenylalanine-derivative hypoglycemic agent

Molecular structures of 10 metabolites, which were isolated from urine (M1-M8) or bile (M9 and M10) after administration of AY4166 (N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine), a novel amino acid derivative with hypoglycemic activity, have been elucidated by mass spectrometry and nuclear magnetic resonance. Four of these (M1, M2, M3 and M8) were determined to be hydroxyl derivatives of AY4166, two (M9 and M10) were carboxylate derivatives via oxidization of M2 and M3, three (M4, M5 and M6) were glucronic acid conjugates and the other (M7) was a dehydro derivative. The estimated structures for M1, M2, M3, M7, M8, M9 and M10 were confirmed by the coincidence of the retention time of HPLC, MS and 1H NMR spectra between the isolated metabolites and authentic synthesized substances. For three glucronic acid conjugates, M4, M5 and M6, structural confirmation was performed by a selective enzymatic digestion with beta-glucronidase. M1 and M2/3 were about 5-6 and 3 times less potent than AY4166, respectively, and M7 was almost as potent as AY4166.

Determination of ascorbic acid and dehydroascorbic acid in juices by high-performance liquid chromatography with electrochemical detection using l-cysteine as precolumn reluctant

Determination of ascorbic acid (AA) and dehydroascorbic acid (DHAA) in juices was performed by high-performance liquid chromatography with electrochemical detection using l-cysteine as precolumn reluctant. This method was suitable for the determination of AA and total AA (AA + DHAA) in juices. The mild reduction of DHAA to AA with l-cysteine took ca. 15 min, and the retention time of AA was ca. 15 min. The detection limit (signal-to-noise ratio = 2) was ca. 0.15 ng. The method was selective and reproducible (relative standard deviation 2.6–3.2% for AA and 2.1–3.2% for total AA). The calibration graph for AA was linear in the range 0.1–10 ng. The recovery of AA was over 90% by the standard addition method.

Determination of ascorbic acid in food by column liquid chromatography with electrochemical detection using eluent for pre-run sample stabilization

Determination of ascorbic acid (AA) in food was performed by column liquid chromatography with electrochemical detection using an eluent (100 mM KH2PO4 (pH 3) with 1 mM ethylenediaminetetraacetic acid disodium dihydrate) for pre-run sample stabilization. The applied potential was set at 400 mV vs. an Ag/AgCl reference electrode. The proposed method was simple, rapid (analysis time: ca. 8 min), sensitive (detection limit: ca. 0.5 ng per injection (20 microliters) at a signal-to-noise ratio of 3), highly selective and reproducible [relative standard deviation: ca. 1.8% (n = 5)]. The calibration graph for AA was linear in the range 0.1-16 ng per injection (20 microliters). Recovery of AA was over 90% by the standard addition method.

Determination of cyanocobalamin in foods by high-performance liquid chromatography with visible detection after solid-phase extraction and membrane filtration for the precolumn separation of lipophilic species

Determination of trace amounts of cyanocobalamin (18 ng/g) in fatty foods was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with visible detection at 550 nm using a membrane filter for the precolumn separation of particulate material. It was found that a membrane filter (HLC-Disk 25, 0.45 μm) was most suitable for the separation of oily particulates. A sample solution was applied to a solid-phase extraction cartridge and then cyanocobalamin was eluted using a 50% aqueous acetonitrile solution followed by HPLC. This method was suitable for the determination of trace amounts of cyanocobalamin in nutrient samples. The proposed method was simple, rapid (extraction time: ca. 12 min, analysis time: ca. 12 min), sensitive (detection limit: ca. 0.15 ng at a signal-to-noise ratio of 3:1), highly selective and reproducible (relative standard deviation: 2.67%) for cyanocobalamin. The calibration graph for cyanocobalamin was linear in the range of 0.1 to 30 ng. Recovery of cyanocobalamin was over 90% by the standard addition method.

Determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine and its metabolites in human plasma and urine by column-switching high-performance liquid chromatography with ultraviolet detection

A simple, rapid and sensitive two column-switching high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (AY4166, I) and its seven metabolites in human plasma and urine. Measurements of I and its metabolites were carried out by two column-switching HPLC, because metabolites were classified into two groups according to their retention times. After purification of plasma samples using solid-phase extraction and direct dilution of urinary samples, I and each metabolite were injected into HPLC. The calibration graphs for plasma and urinary samples were linear in the ranges 0.1 to 10 microg ml(-1) and 0.5 to 50 microg ml(-1), respectively. Recoveries of I and its seven metabolites were over 88% by the standard addition method and the relative standard deviations of I and its metabolites were 1-6%.

Determination of amino acids in human plasma by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination.

Amino acids in human plasma were determined by liquid chromatography with postcolumn ninhydrin derivatization using a hydroxyapatite cartridge for precolumn deproteination. S-Carboxymethyl-L-cysteine, D-phenylglycine and S-aminoethyl-L-cysteine were found to be suitable internal standards. The proposed method is simple, rapid (deproteination time less than 1 min) and reproducible [relative standard deviation below 3% except for low-level aspartic acid (n = 3)]. The average recovery of 25 amino acids was above 90%. The elution time of amino acids in human plasma was approximately 2 h. Protein binding of tryptophan was also determined by the proposed method. The analytical data for amino acids in human plasma deproteinated using the proposed and published methods (5-sulphosalicylic acid and ethanol) were compared.

Determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine in human plasma by solid-phase extraction and column-switching high-performance liquid chromatography with ultraviolet detection

A column-switching high-performance liquid chromatography method with ultraviolet detection at 210 nm has been developed for the determination of N-(trans-4-isopropylcyclohexylcarbonyl)-D-phenylalanine (AY4166, I) in human plasma. Plasma samples were prepared by solid-phase extraction with Sep-Pak Light tC18, followed by HPLC. The calibration graph for I was linear in the range 0.1-20 micrograms/ml. The limit of quantitation of I, in plasma, was 0.05 microgram/ml. The recovery of spiked I (0.5 microgram/ml) to drug-free plasma was over 92% and the relative standard deviation of spiked I (0.5 microgram/ml) compared to drug-free plasma was 4.3% (n = 8).

Determination of ascorbic acid in human plasma by high-performance liquid chromatography with electrochemical detection using a hydroxyapatite cartridge for precolumn deproteinization

Ascorbic acid (AA) was determined in human plasma by high-performance liquid chromatography with electrochemical detection using a hydroxyapatite cartridge for plasma deproteinization. The proposed method is simple, rapid (deproteinization time, within 1 min; analysis time, ca. 15 min), sensitive [detection limit, ca. 240 ng/ml plasma (at a signal-to-noise ratio of 2:1)], highly selective and reproducible [relative standard deviation, ca. 2.8% (n = 3)]. The calibration graph for AA was linear in the range 0.1-10 ng per injection (20 microliters). The recovery of AA was over 90% by the standard addition method.

Novel precolumn deproteinization method using a hydroxyapatite cartridge for the determination of theophylline and diazepam in human plasma by high-performance liquid chromatography with ultraviolet detection

Abstract The deproteinization of human plasma was carried out using a hydroxy apatite cartridge as a precolumn. After human plasma had been passed through the cartridge followed by suitable elution, protein-free eluate was obtained within only 1 min without the need for centrifugation. Deproteinization was evaluated by the determination of albumin, γ-globulin and transferrin in the eluate by high-performance liquid chromatography (HPLC) with UV detection. Determination of theophylline and diazepam in human plasma was performed by HPLC with UV detection. The proposed method was suitable for the determination of these two drugs in human plasma, because it is simple and rapid (retention time of each drugs ≈ 15 min) and microamounts of sample are required (50–100 μl). The calibration graphs for theophylline and diazepam were linear in the range 0.1–10 μg and 0.1–65 ng, respectively. Recoveries of both drugs were over 90% by the standard addition method.

Determination of a new calcium antagonist and its main metabolite in plasma by thermospray liquid chromatography-mass spectrometry

A highly sensitive thermospray liquid chromatographic-mass spectrometric method has been developed for the simultaneous determination of FRC-8653 (I), a new calcium antagonist, and its main metabolite (M-4) in plasma. A deuterated analogue of I was added to the plasma as the internal standard. After the purification and concentration of the plasma sample on bonded-phase disposable columns, the extract was injected into the thermospray liquid chromatograph and analysed by selected-ion monitoring mass spectrometry. The calibration curves obtained were linear over the concentration range 0.5-100 ng/ml. The limits of quantification are 0.5 ng/ml for I and 1 ng/ml for M-4 in plasma, which are sufficient to evaluate plasma concentrations after oral administration to rats.