A novel fluorescent probe, 1-[1,2,5,6-dibenzocarbazol-9-yl]propan-2-yl chloroformate
(DBCPC-Cl) has been designed and synthesized for amine labeling in HPLC. Using 12 aliphatic
amines as the models, the derivatization conditions were optimized. In 0.2 mol=L borate buffer
(pH 9.0), amines reacted with DBCPC-Cl at 30C to form the derivatives in 3 min, and the derivatives
were stable enough to be efficiently analyzed by HPLC. The separation of these amine derivatives
was achieved with a C8 column and gradient elution by using 30% acetonitrile (containing
20mmol=L formic acid) and 100% acetonitrile, and online APCI=MS identification of the derivatives
was carried out in positive-ion mode. The fluorescence responses for DBCPC-derivatives were
higher relative to those obtained using previously reported reagents. With fluorescence detection at
an emission wavelength of 390nm and an excitation wavelength of 300nm, the detection limits
of aliphatic amines were 0.3 – 5.1 fmol (at a signal-to-noise ratio of 3:1). The relative standard
deviations for within-day determination (n¼12) were 0.051–0.079% for retention time and
0.84–1.12% for peak area for the tested aliphatic amines. The mean intra- and inter-assay
precision for all amines levels were <2.52% and 3.74%, respectively. The mean recoveries ranged
from 86.9 to 104.7%. Excellent linear responses were observed with coefficients >0.9991.
The research has been published on Journal of Liquid Chromatography & Related Technologies, 33:390–404, 2010
Zhiwei Sun,1,3 Lian Xia,1,2,3 Yourui Suo,1 and Jinmao You1,2