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 ACTH 4–9 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°C for 30 min. After cooling, the mixture was seperated by a reversed‐phase HPLC. Then DBD‐ebiratide was monitored at 580 nm with the excitation at 440 nm. The calibration curve was liner up to 10 pmol on column ( r=0.999 ) with the detection limit of 0.25 pmol (signal‐to‐noise=3).