Ken-ichi Mawatari

Determination of quinolinic acid in human urine by liquid chromatography with fluorimetric detection

Abstract A system has been developed for the determination of quinolinic acid in human urine using liquid chromatography with fluorimetric detection. The compound is separated by reversed-phase chromatography using a 35 mM potassium dihydrogen-phosphate solution (adjusted to pH 3.8 with 0.2 M citric acid) containing 350 mM hydrogen peroxide and 0.05 mM tetramethylammonium hydroxide as a mobile phase. The compound in the column effluent is irradiated with ultraviolet light to produce fluorescence. This fluorescence is monitored with excitation at 326 nm and emission at 380 nm. The calibration graph for quinolinic acid is linear over the range 0.36–68.8 nmol ml−1 upon injecting 10 μl of the standard solution. Pretreatment of urine was achieved by dilution and filtration only. The mean recovery of quinolinic acid from urine is more than 95%.

Simultaneous determination of nucleosides and nucleotides in dietary foods and beverages using ion-pairing liquid chromatography–electrospray ionization-mass spectrometry

A method using ion-pairing liquid chromatography-electrospray ionization (ESI)-mass spectrometry (MS) was developed for the simultaneous determination of 23 types of purine or pyrimidine nucleosides and nucleotides in dietary foods and beverages. Dihexylammonium acetate (DHAA) was used as an ion-pairing agent and an ultra performance liquid chromatography (UPLC) system with a reversed-phase column and a gradient program was employed for the separation of nucleosides and nucleotides. Positive-ion ESI-MS was applied for the detection of nucleosides, and negative-ion ESI-MS was used for nucleotides. Lower limits of quantitation ranged from 0.02 micromol/L (UMP and AMP) to 1.3 micromol/L (CDP). The present method was validated, and sufficient reproducibility and accuracy was obtained for the quantitative measurement of the 23 types of nucleosides and nucleotides. The method was subsequently applied to their determination in a range of Japanese foods and beverages that are considered to contain significant amounts of umami flavor compounds. Because dietary purine nucleosides and nucleotides are known to be related to hyperuricemia and gout, the determination of their concentrations in dietary foods is useful for both evaluating umami flavor and assessing the effects of dietary food on purine metabolism.

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Analysis of urinary calculi obtained from a patient with idiopathic hypouricemia using micro area x-ray diffractometry and LC-MS

Urolithiasis is a common complication in patients with hypouricemia. Using a microarea x-ray diffractometer and nanoflow liquid chromatography-mass spectrometry (LC-MS) following SDS-polyacrylamide gel electrophoresis (PAGE), recurrent urinary calculi complicating a hypouricemic patient were analyzed. Analysis with the microarea x-ray diffractometer showed that one of the calculi was composed of calcium oxalate monohydrate and hydroxyapatite. The other was found to be formed from calcium oxalate dihydrate. After determination with LC-MS, both were found to contain uromodulin, albumin, osteopontin, protein Z, and defensins. Lysozyme and calgranulin A were also identified in these calculi. Defensins, which were antimicrobial peptides, and lysozyme, a mucopeptide glycohydrolase, were identified as new organic components of urinary stones. The role of these proteins in the process of urolithiasis is of particular interest.

Determination of disodium cromoglycate in human urine by high-performance liquid chromatography with post-column photoirradiation-fluorescence detection

For the determination of disodium cromoglycate in urine, a fluorimetric method using HPLC post-column photoirradiation has been developed. The mobile phase consisted of a 35 mmol l-1 phosphate buffer (pH 8)-methanol (7 + 3, %v/v) containing 75 mmol l-1 hydrogen peroxide and 20 mmol l-1 18-crown-6. The 18-crown-6 was used for separation adjustment of the disodium cromoglycate in the urine sample. Photoirradiation was carried out in tubing wound around a germicidal light in a reactor equipped with an air-cooling fan. The fluorescence was monitored with excitation at 325 nm and emission at 448 nm. The calibration graph for disodium cromoglycate was linear over the range 38-2340 ng ml-1 using an injection volume of 100 microliters. The pretreatment of the urine samples consisted of diluting and filtering steps. The mean recovery of disodium cromoglycate from urine was 99.1 +/- 2.4% (n = 6).

Analysis of Purine in Purine-Rich Cauliflower

Purine is a general term for purine nucleotides, nucleosides, bases, and nucleic acid. The amount of purine nucleotides, nucleosides, and bases in purine-rich cauliflower was determined with the use of LC-MS and HPLC, and the ratio of these molecules were compared with in raw and in heated condition. Total purine content of raw and heated cauliflower was 42.6 and 43.2 mg/100 g, respectively. Nucleotide content was increased from 0.02 to 50.8 μmol/100 g, and nucleoside content was decreased from 12.4 to 7.7 μmol/100 g, by heating.

Purine contents of soybean-derived foods and selected Japanese vegetables and mushrooms.

Purine contents of soybean-derived food and various other Japanese foods were quantitatively determined by high-performance liquid chromatography (HPLC). Purine contents were as follows: soybean-derived foods, 21.9–172.5 mg/100 g or 100 mL; Japanese vegetables, 2.3–171.8 mg/100 g; Japanese mushrooms, 9.5–142.3 mg/100 g. Since purine levels in these foods did not exceed 200 mg/100 g, we recommend that eating of them should be adopted and good dietary habits followed.

Simultaneous determination of nicotine and cotinine in serum using high-performance liquid chromatography with fluorometric detection and postcolumn UV-photoirradiation system

A simple and rapid method for the simultaneous determination of serum nicotine and cotinine using high-performance liquid chromatography (HPLC)-fluorometric detection with a postcolumn ultraviolet-photoirradiation system was developed. Analytes were extracted from alkalinized human serum via liquid-liquid extraction using chloroform. The organic phase was back-extracted with the acidified aqueous phase, and the analytes were directly injected into an ion-pair reversed-phase HPLC system. 6-Aminoquinoline was used as an internal standard. Nicotine, cotinine, and 6-aminoquinoline were separated within 14min. The extraction efficiency of nicotine and cotinine was greater than 91%. The linear range was 0.30-1000ng for nicotine and 0.06-1000ng for cotinine. In serum samples from smokers, the concentrations of nicotine and cotinine were 8-15ng/mL and 156-372ng/mL, respectively.

Fluorescence enhancement by hydroperoxides based on a change in the intramolecular charge transfer character of benzofurazan

Strong fluorescence signals were observed after the reaction of novel reagents with hydroperoxides.

Fluorimetric determination of nicorandil in human plasma by a high-performance liquid chromatographic-postcolumn ultraviolet detection system equipped with on-line back-pressure tubing

Abstract For the determination of nicorandil in plasma, a fluorometric technique using HPLC-postcolumn UV detection has been developed. The chromatographic system consisted of a single pump, photoreactor and on-line back-pressure tubing. The system was suitable for the separation of nicorandil under the present reaction conditions. The calibration graph was linear over the range 6.5–1170 ng ml−1 using an injected volume of 100 μ1. The pretreatment of the plasma samples consisted only of deproteinizing steps by adding perchloric acid. The mean recovery from plasma was 90.2%.