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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
Author(s) -
Hamachi Yozo,
Tsujiyama Tsuyosi,
Nakashima Kenichiro,
Akiyama Shuzo
Publication year - 1995
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.1130090505
Subject(s) - chemistry , chromatography , reagent , high performance liquid chromatography , detection limit , fluorescence , acetonitrile , calibration curve , reversed phase chromatography , organic chemistry , physics , quantum mechanics
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).