Determination of amines using 2-(11 H-benzo[a]carbazol-11-yl) ethyl chloroformate (BCEC-Cl) as labeling reagent by HPLC with fluorescence detection and identification with APCI/MS

Abstract

A pre-column derivatization method for the sensitive determination of amines using a labeling reagent 2-(11 H-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-(11 H-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 2O–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 0.50 pmol injection, at a signal-to-noise ratio of 3, were 1.77–14.4 fmol. 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.