Analytical Sciences

Fluorometric Determination of Khellinin Human Urine and Serum by High-Performance Liquid Chromatography UsingPostcolumn Photoirradiation

Ken-ichi MAWATARI, Satoshi MASHIKO,Mitsuo WATANABE, and Kazuya NAKAGOMI

Department of Analytical Chemistry,Faculty of Pharmacutical Sciences, Teikyo University, Sagamiko, Kanagawa199-0195, Japan

For the determination of khellin in urineand serum, fluorometry using HPLC-postcolumn photoirradiation has beendeveloped. Khellin and visnagin of similar structure were separated on acolumn of Capcell Pak C₈. The mobile phase consisted of40%(v/v) ethanol containing 75 mmol l^-1H₂O₂. The postcolumn reagent, 70 mmol l^-1KH₂PO₄-NaOH buffer (pH 12.7) containing 50%(v/v)ethanol, were mixed with the mobile phase, which was irradiated withultraviolet light to induce fluorescence. The fluorescence was monitoredwith excitation at 378 nm and emission at 480 nm. The calibration graphfor khellin was linear over the range of 65 - 2620 ng ml^-1 usingan injection volume of 20 µl. The pretreatment of the urine or serumsamples consisted of diluting steps or deproteinizing steps usingperchloric acid, respectively.

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