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).
Hamachi
Y
Y
Laboratory of Analytical Research, Hoechst Japan Limited, Kawagoe, Japan.
Tsujiyama
T
T
Nakashima
K
K
Akiyama
S
S
D000324:Adrenocorticotropic Hormone / Q000031:analogs & derivatives* / Q000097:blood; D000595:Amino Acid Sequence; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010080:Oxazoles; D010446:Peptide Fragments / Q000097:blood*; D051381:Rats; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013439:Sulfhydryl Reagents; D013449:Sulfonamides; D013696:Temperature
D005456:Fluorescent Dyes; D010080:Oxazoles; D010446:Peptide Fragments; D013439:Sulfhydryl Reagents; D013449:Sulfonamides; D000324:Adrenocorticotropic Hormone; C060548:4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole; C057139:ebiratide