Hiroko Kawamoto

Determination of hydrogen peroxide by micro-flow injection-chemiluminescence using a coupled flow cell reactor chemiluminometer

A novel flow cell reactor was developed for micro-flow injection determination of hydrogen peroxide (H(2)O(2)) using horseradish peroxide (HRP)-catalysed luminol chemiluminescence. The newly developed flow cell reactor for a chemiluminometer allowed mixing of the chemiluminescent reagents in front of a photomultiplier for maximum detection of the emitted light. The rapid mixing allowed a decrease in the flow rate of the pump to 0.1-0.01 mL/min, resulting in increased sensitivity of detection of light. The flow cell reactor was made by packing HRP-immobilized gels into a flow cell (Teflon tube; 6 cm x 0.98 mm i.d.) located in the cell holder of a chemiluminometer (flow-through type). The HRP-immobilized gels were made by immobilizing HRP onto the Chitopearl gel by the periodate method. H(2)O(2) specimens (50 microL) were injected into a stream of water delivered at a flow rate of 0.1 mL/min and mixed with a luminol solution (0.56 mmol/L in Tricine buffer, pH 9.2) delivered at 0.1 mL/min in the flow cell reactor. Within-run reproducibility of the assay of H(2)O(2) was 2.4% (4.85 micromol/L; flow rate 0.1 mL/min, injection interval 10 min). The reproducibility of the H(2)O(2) assay was influenced by the flow rates and the injection intervals of the H(2)O(2) specimens. As the flow rates decreased, both the light intensity and the light duration increased. Optimal light intensity was obtained at a luminol concentration of 3-8 mmol/L, but 0.56 mmol/L was sufficient for assay of H(2)O(2) in clinical specimens. At a luminol concentration of 0.56 mmol/L, the regression equation of the standard curve for H(2)O(2) (0-9.7 micromol/L) was Y = 27.5 X(2) + 394 X + 58.9 (Y = light intensity; X = concentration of H(2)O(2)) and the detection limit of H(2)O(2) was 0.2 micromol/L. This method was used to assay glucose (2.7-16.7 mmol/L) based on a glucose oxidase (20 U/mL, pH 7.4) reaction. The standard curve for glucose was Y = 167 X(2) - 351 X + 1484 (Y = light intensity; X = glucose). The within-run reproducibility for an aqueous glucose standard (2.7 mmol/L) and a control serum (glucose, 5 mmol/L) was 4.48% (n = 5) and 5.70% (n = 9), respectively.

Fluorimetric determination of serum 11-hydroxysteroids. Calculative correction of deviations in complex fluorescence spectra

Abstract Fluorimetry of serum 11-hydroxycorticosteroids was accompanied by contamination due to nonspecific fluorescence. A spectro-fluorometrical method was proposed for the correction of the contamination.

Total free catecholamines assay by identification of its two functional groups and micro-flow injection chemiluminescence.

We have developed a novel method of assaying total free catecholamines using sulphuric acid-derivatized beads for extracting and identifying catecholamine (CA) on the surface, and assaying the peroxide produced from CA by chemiluminescence (CL). Current assay methods for CA by electrochemical determination, fluorescence and chemiluminescence need a time-consuming separation by high-performance liquid chromatography. We eliminated this separation step by identifying the two functional groups of CA using a derivatized bead and this resulted in a highly specific CA assay. The principle is as follows: the amino group of CA was trapped by ion binding with a sulphuric acid derivative immobilized on a bead, and the diol of the CA bound to the bead was converted to peroxide with imidazole under alkaline conditions. The peroxide produced was assayed by microflow injection-horseradish peroxidase-catalysed luminol chemiluminescence. We synthesized three types of sulphuric acid-derivative immobilized beads (6.5 mm i.d.). The types of immobilized sulphuric acid derivative used were straight-chain, branched chain and benzenesulphonic, respectively. The order of the three types of beads for extracting CA was: bezenesulphonic type > branched type > straight-chain type. The optimal incubation time for generating peroxide was 30 min. The peroxide generated in the reaction solution was stable with within-run reproducibility of CV 5. 7% after incubation for 80 min. The regression equation of a standard curve for dopamine was Y = 12.8 X(2) + 476X - 373 (where Y = light intensity (RLU), X = concentration of dopamine (micromol/L)). The minimum detection limit of dopamine was 0.1 micromol/L, and the within-run reproducibility of dopamine (10.5 micromol/L) was CV 4.7% (n = 5). This method is applicable to assay of total free CA without use of HPLC.

A new method for estimation of corticosteroid-binding globulin with hydrophobic resin

Abstract A new method is described for the estimation of corticosteroid-binding globulin by means of the hydrophobia resin adsorption method. Corticosteroid-binding globulin in the serum is saturated by incubation with cortisol, unbound and albumin-bound cortisol is removed by shaking with the resin, and cortisol remaining in the supernatant fluid is measured by fluorometry, its represents the cortisol-binding capacity of corticosteroid-binding globulin. Normal range of cortisol-binding capacity was 0.154–0.222 μg/ml at 15°.