Determination of amines using 2-(11H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS.
A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11H-benzo[a]-carbazol-11-yl) ethyl chloroformate (BCEC-Cl) followed by high-performance liquid chromatography with fluorescence detection has been developed. Identification of derivatives was carried out by LC/APCI/MS in positive-ion mode. The chromophore of 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) reagent was replaced by 2-(11H-benzo[a]-carbazol-11-yl) ethyl functional group, which resulted in a sensitive fluorescence derivatizing reagent BCEC-Cl. BCEC-Cl could easily and quickly label amines. Derivatives were stable enough to be efficiently analyzed by HPLC and showed an intense protonated molecular ion corresponding m/z [M+H](+) under APCI/MS in positive-ion mode. The collision-induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 261.8 and m/z 243.8 corresponding to the cleavages of CH(2)O-CO and CH(2)-OCO bonds. Studies on derivatization demonstrated excellent derivative yields over the pH 9.0-10.0. Maximal yields close to 100% were observed with three- to four-fold molar reagent excess. In addition, the detection responses for BCEC-derivatives were compared to those obtained using 1,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC-Cl) and 9-fluorenyl methylchloroformate (FMOC-Cl) as labeling reagents. The ratios I(BCEC)/I(BCEOC)=1.94-2.17 and I(BCEC)/I(FMOC)=1.04-2.19 for fluorescent (FL) responses (here, I was relative fluorescence intensity). Separation of the derivatized amines had been optimized on reversed-phase Eclipse XDB-C(8) column. Detection limits calculated from 0.50pmol injection, at a signal-to-noise ratio of 3, were 1.77-14.4fmol. The relative standard deviations for within-day determination (n=11) were 1.84-2.89% for the tested amines. The mean intra- and inter-assay precision for all amines levels were <3.64% and 2.52%, respectively. The mean recoveries ranged from 96.6% to 107.1% with their standard deviations in the range of 0.8-2.7. Excellent linear responses were observed with coefficients of >0.9996.
You
Jinmao
J
Northwest Plateau Institute of Biology, Chinese Academy of Sciences, Xining 810001, PR China; College of Chemistry Science, Qufu Normal University, Qufu Shandong 273165, PR China.
Zhao
Wenchen
W
Liu
Lingjun
L
Zhao
Xianen
X
Suo
Yourui
Y
Wang
Honglun
H
Li
Yulin
Y
Ding
Chenxu
C