Hiroshi Iwase

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.

Ultramicrodetermination of cyanocobalamin in elemental diet by solid-phase extraction and high-performance liquid chromatography with visible detection.

The ultramicrodetermination of cyanocobalamin (9 ng/g) in elemental diet containing 46 kinds of compounds, with concentrations at least 50-10(6) times higher than that of cyanocobalamin, was performed by Sep-Pak C(18) purification and concentration of cyanocobalamin followed by HPLC with detection at 550 nm. The method is simple, rapid, sensitive and reproducible. The calibration graph was linear in the range of 0-0.2 micrograms. The recovery of cyanocobalamin was over 95% by the standard addition method. There was good agreement between the cyanocobalamin concentrations indicated and found.

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.

Routine high-performance liquid chromatographic determination of ascorbic acid in foods using L-methionine for the pre-analysis sample stabilization.

The possibility of use of phosphoric acid (0.2% v/v, pH 2.1) in the mobile phase and co-existing compounds present in foods as the dissolving agent for the pre-analysis sample stabilization were examined for the routine determination of ascorbic acid (AA) in foods by high-performance liquid chromatography (HPLC) with electrochemical detection (ED). The applied potential was set at 400 mV versus an Ag/AgCl reference electrode. It was demonstrated that 0.2% v/v phosphoric acid was the useful mobile phase and L-methionine was the most effective dissolving agent for the pre-run sample stabilization of AA in foods after comparison with other amino acids and water-soluble vitamins. The proposed method was simple, rapid (retention time @ ca. 4 min), sensitive (detection limit: ca. 0.1 ng per injection (5 microl) at a signal-to-noise ratio of 3), highly selective and reproducible (relative standard deviation (R.S.D.); 2.5% (n=7), between-day R.S.D.: 3.7% (5 days)). The calibration graph of AA was linear in the range of 0.1-12.5 ng per injection (5 microl). Recovery of AA was over 90% by the standard addition method. Relationship between structure of compounds and the stability of AA was also examined.

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 ascorbic acid in elemental diet by high-performance liquid chromatography with electrochemical detection

Determination of ascorbic acid in a multi-component elemental diet was performed by high-performance liquid chromatography with electrochemical detection. This method is suitable for the routine determination of ascorbic acid in elemental diet because it is simple, rapid, sensitive, highly selective and reproducible. The calibration graph of ascorbic acid was linear in the range 0-1.0 micrograms. The recovery of ascorbic acid was over 95% by the standard addition method. There was good agreement between the concentrations of ascorbic acid stated and found.

Determination of vitamin K1 in emulsified nutritional supplements by solid-phase extraction and high-performance liquid chromatography with postcolumn reduction on a platinum catalyst and fluorescece detection

Determination of small amounts of vitamin K1 (0.8 microg/g) in nutritional supplements with high fat content (20 mg/g) was performed by solid-phase extraction and high-performance liquid chromatography (HPLC) with fluorescence detection after reduction on a platinum oxide catalyst. The concentration ratio of plant oils to vitamin K1 (0.8 microg/g) was about 25,000:1. A sample solution was applied to a solid-phase extraction cartridge and vitamin K1 was eluted with ethanol, followed by HPLC. The proposed method was simple, rapid (analysis time: ca. 12 min), sensitive [detection limit: ca. 0.1 pg per injection (100 microl) at a signal-to-noise ratio of 3:1], highly selective and reproducible [relative standard deviation: ca. 1.3%. (n=5)]. The calibration graph of vitamin K1 was linear in the range of 0-2 pg per injection (100 microl). Recovery of vitamin K1 was over 90% by the standard addition method.

Determination of vitamin D2 in emulsified nutritional supplements by solid-phase extraction and column-switching high-performance liquid chromatography with UV detection

This paper deals with a method for solid-phase extraction of trace amounts of vitamin D2 (VD2, 19 ng/g) from emulsified nutritional supplements, which contain 50 kinds of compounds, followed by column-switching high-performance liquid chromatography (HPLC) with UV detection at 265 nm. VD2 is present at 1000-20,000,000 times lower concentration than other components. Bond Elut C18 cartridge was chosen as for the emulsified nutritional supplements after comparison with eight other types. A sample solution was applied to the solid-phase extraction cartridge and VD2 was eluted by methanol followed by HPLC. The effects of sample pH, eluent composition and eluate volume on the retention and elution of VD2 on Bond Elut C18 cartridge were examined. The resulting method was simple, rapid (analysis time: approximately 20 min), sensitive (detection limit: approximately 0.1 ng per injection (200 microl) at a signal-to-noise ratio 3:1), and reproducible (relative standard deviation: approximately 6.2%, n=5). The calibration graph for VD2 was linear in the range of 0.1-3 ng per injection (200 microl). Recovery of VD2 was approximately 80% by the standard addition method.

Determination of tocopherol acetate in emulsified nutritional supplements by solid-phase extraction and high-performance liquid chromatography with fluorescence detection

The present paper deals with a method of solid-phase extraction of tocopherol acetate (TA, 49.6 microg/g) from emulsified nutritional supplements, which contains 50 kinds of compounds, followed by high-performance liquid chromatography (HPLC) with fluorescence detection The TA concentration is 5 to approximately 100,000 times lower than that of other compounds in the samples. Measuring the loading capacity of the larger amounts of vegetable oil onto the Bond Elut C18 cartridge was examined for the complete retention of smaller level of TA. A sample solution was applied to a solid-phase extraction cartridge and then TA was eluted by acetonitrile followed by HPLC. This method was suitable for the determination of TA in emulsified nutritional supplements. The proposed method was simple, rapid (analysis time: ca. 15 min), sensitive [detection limit: ca. 0.1 ng per injection (100 microl) at a signal-to-noise ratio of 3:1], and reproducible (relative standard deviation: ca. 2.5% (n=5)). The calibration graph of TA was linear in the range of 0.1 to 100 ng per injection (100 microl). Recovery of TA was over 90% by the standard addition method.

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.