Fluorometric Determination of Methotrexate in Serum by High- Performance Liquid Chromatography Using In-Line Oxidation with Hydrogen Peroxide

Hiroaki KUBO; Yoshikazu UMIGUCHI; Mariko FUKUMOTO; Toshio KINOSHITA

doi:10.2116/analsci.8.789

Hiroaki KUBO, Yoshikazu UMIGUCHI, Mariko FUKUMOTO, Toshio KINOSHITA

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Keywords: High-performance liquid chromatography, methotrexate, serum, fluorometric detection, hydrogen peroxide, in-line oxidation, fluorescence polarization immunoassay

JOURNAL FREE ACCESS

1992 Volume 8 Issue 6 Pages 789-792

DOI https://doi.org/10.2116/analsci.8.789

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Published: December 10, 1992 Received: June 30, 1992 Available on J-STAGE: June 30, 2006 Accepted: September 11, 1992 Advance online publication: - Revised: -
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Date of correction: June 30, 2006 Reason for correction: - Correction: ABSTRACT Details: Wrong : A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25mM phosphate buffer) containing 25mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379nm and Em 457nm) via in-line oxidation at high temperature (160°C). The calibration curve could be made linear over the range 1×10^-6-1×10^-5M by injecting a volume of 20μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2×10^-8M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.
Right : A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25mM phosphate buffer) containing 25mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379nm and Em 457nm) via in-line oxidation at high temperature (160°C). The calibration curve could be made linear over the range 1×10^-6-1×10^-5M by injecting a volume of 20μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2×10^-8M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.

Date of correction: June 30, 2006 Reason for correction: - Correction: PDF FILE Details: -

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Abstract

A fluorometric method has been developed for the determination of methotrexate in serum by high-performance liquid chromatography. Methotrexate after deproteinization with 2M perchloric acid was separated by reversed-phase chromatography with a neutral mobile phase (pH 7.0, 8% acetonitrile in 25mM phosphate buffer) containing 25mM hydrogen peroxide as a fluorogenic reagent, and was fluorometrically detected (Ex 379nm and Em 457nm) via in-line oxidation at high temperature (160°C). The calibration curve could be made linear over the range 1×10^-6-1×10^-5M by injecting a volume of 20μl of deproteinized serum. The detection limit (signal-to-noise ratio=3) for methotrexate in serum was 2×10^-8M using a 50-μl aliquot of deproteinized serum. Comparison with the fluorescence polarization immunoassay gave a good correlation coefficient of 0.949.

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