Direct Determination of Platelet-Produced Thromboxane B₂ in Human Serum by Column-Switching High-Performance Liquid Chromatography with Fluorescence Detection

抄録

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.