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

Abstract

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).

Fluorogenic and fluorescent labeling reagents with a benzofurazan skeleton

“…The detection limits for DBD-amino acids derivatives were 0.11-0.79 pmol (S/N = 3) using HPLC (Toyo'oka et al, 1989d). DBD-F as a pre-column fluoregenic reagent was applied to the determination of N-terminal prolyl peptides (Toyo'oka et al, 1994a), salmon calcitonin (Fukuda et al, 1995) and ebiratide (Hamachi et al, 1995). Since DBD-amine derivatives are sensitively detected by peroxyoxalate chemiluminescence detection, DBD-F was used for the determination of b-blocker metoprolol , epinephrine (Uzu et al, 1991b), methamphetamine and its related compounds and ebiratide (Hamachi et al, 1997) with this detection method.…”

Analytical Studies on the Development of High-Performance Liquid Chromatographic Methods with Fluorescence or Chemiluminescence Detections and Their Practical Applications

“…19 Moreover, ebiratide, one of the synthetic analogues of ACTH4-9 for Alzheimer disease could also be determined and its time course in rat plasma could be traced with the lower levels of sub-pmol/mL. 20,21 2·2 Lophine derivatives [22][23][24][25][26][27][28][29][30][31][32][33][34] Lophine derivatives are well-known potential fluorescent or chemiluminescent compounds. Therefore, a variety of lophine derivatives were synthesized as shown in Fig.…”