Direct determination of platelet-produced thromboxane B-2 in human serum by column-switching high-performance liquid chromatography with fluorescencedetection

A high-performance liquid chromatographic method for the direct determinati on of platelet-produced thromboxane B-2 in human serum is described. The me thod is based on direct derivatization of the thromboxane B-2 in human seru m with 6,7-dimethoxy-1 -methyl-2(1H)-quinoxalinone-3-propionyl carboxylic a cid hydrazide. The derivatization reaction proceeds in aqueous solution (or serum sample) in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopr opyl)carbodiimide at 37 degrees C. The resulting fluorescent derivative of thromboxane B-2 is separated by column-switching chromatography using a fir st column (YMC-Pack C-4) 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 B-2 is 10 pmol (3.7 ng)/ml serum. The present method is highly sensitive and si mple without any clean-up, such as in conventional liquid-liquid and solid- phase extraction.