High-performance liquid chromatography with fluorescence detection of ebiratide using 4-(N,N-dimethylamino-sulphonyl)-7-fluoro-2,1,3-benzoxadiazole as a fluorogenic reagent.

A sensitive high-performance liquid chromatography (HPLC) method with fluorescence detection has been developed and applied to the determination of ACTH4-9 analogue (ebiratide). 4-(N,N-Dimethylamino-sulphonyl)-7-fluoro-2,1,3-benzoxadiazole (DBD-F) was used as a fluorogenic labelling reagent. Ebiratide in 0.1 M borate buffer (pH 9.0) was reacted with DBD-F in acetonitrile solution at 50 degrees C for 30 min. After cooling, the mixture was separated by a reversed-phase HPLC. Then DBD-ebiratide was monitored at 580 nm with the excitation at 440 nm. The calibration curve was linear up to 10 pmol on column (r = 0.999) with the detection limit of 0.25 pmol (signal-to-noise = 3).