Abstract − Analytical Sciences, 16(1), 45 (2000).
Direct Determination of Platelet-Produced Thromboxane B2 in Human Serum by Column-Switching High-Performance Liquid Chromatography with Fluorescence Detection
Tetsuharu IWATA, Kazuhito HIROTA, and Masatoshi YAMAGUCHI
Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Jonan, Fukuoka 814-0180, Japan
A high-performance liquid chromatographic method for the direct determination of platelet-produced thromboxane B2 in human serum is described. The method is based on direct derivatization of the thromboxane B2 in human serum with 6,7- dimethoxy-1-methyl-2(1H)-quinoxalinone-3-propionyl carboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution (or serum sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 37°C. The resulting fluorescent derivative of thromboxane B2 is separated by column-switching chromatography using a first column (YMC-Pack C4) for the trap and clean-up of the derivative and a second column (YMC-Pack Ph) for complete separation of the derivative. The derivative is detected spectrofluorometrically at 445 nm with excitation at 367 nm. The detection limit (signal to noise ratio=3) for thromboxane B2 is 10 pmol (3.7 ng)/ml serum. The present method is highly sensitive and simple without any clean-up, such as in conventional liquid-liquid and solid-phase extraction.
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