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Updated: Sep 21, 2026

Post Column Derivatization Using Reaction Flow High Performance Liquid Chromatography Columns
Published on: April 26, 2016
Fluorometric determination of khellin in human urine and serum by high-performance liquid chromatography using
Ken-ichi Mawatari1, Satoshi Mashiko, Mitsuo Watanabe
1Department of Analytical Chemistry, Faculty of Pharmacutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan.
Abstract:
For the determination of khellin in urine and serum, fluorometry using HPLC-postcolumn photoirradiation has been developed. Khellin and visnagin of similar structure were separated on a column of Capcell Pak C8. The mobile phase consisted of 40%(v/v) ethanol containing 75 mmol l(-1) H2O2. The postcolumn reagent, 70 mmol l(-1) KH2PO4-NaOH buffer (pH 12.7) containing 50%(v/v) ethanol, were mixed with the mobile phase, which was irradiated with ultraviolet light to induce fluorescence. The fluorescence was monitored with excitation at 378 nm and emission at 480 nm. The calibration graph for khellin was linear over the range of 65 - 2620 ng ml(-1) using an injection volume of 20 microl. The pretreatment of the urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively.
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