Determination of Aliphatic Amines from Soil and Wastewater of a Paper Mill by Pre‐Column Derivatization using HPLC and Tandem Mass Spectrometry (HPLC‐MS/MS)

Abstract

A pre‐column derivatization method for the sensitive determination of aliphatic amines using the labeling reagent 1,2‐benzo‐3,4‐dihydrocarbazole‐9‐ethyl chloroformate (BCEOC) followed by HPLC with fluorescence detection and APCI/MS identification in positive‐ion mode has been developed. The chromophore of 2‐(9‐carbazole)‐ethyl chloroformate (CEOC) reagent was replaced by the 1,2‐benzo‐3,4‐dihydrocarbazole functional group, which resulted in a sensitive fluorescence derivatizing reagent, BCEOC, that 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]+ with APCI/MS in positive‐ion mode. The collision induced dissociation of the protonated molecular ion formed characteristic fragment ions at m/z 264.1, m/z 246.0 and m/z 218.1, corresponding to the cleavages of CH2CH2O‐CO, CH2CH2‐OCO, and N‐CH2CH2O bonds. Studies on derivatization conditions demonstrated that excellent derivatization yields close to 100% were observed with a 3 to 4‐fold molar reagent excess in acetonitrile solvent, in the presence of borate buffer (pH 9.0) at 40°C for 10 min. In addition, the detection responses for BCEOC derivatives were compared with those obtained with CEOC and FMOC as labeling reagents. The ratios IBCEOC/ICEOC and IBCEOC/IFMOC were, respectively, 1.40–2.76 and 1.36–2.92 for fluorescence responses (here, I was the relative fluorescence intensity). Separation of the amine derivatives had been optimized on an Eclipse XDB‐C8 column. Detection limits calculated from an 0.10 pmol injection, at a signal‐to‐noise ratio of 3, were 18.65–38.82 fmol (injection volume 10 µL) for fluorescence detection. The relative standard deviations for intraday determination (n=6) of standard amine derivatives (50 pmol) were 0.0063–0.037% for retention times and 3.36‐6.93% for peak areas. The mean intra‐ and inter‐assay precision for all amines were <5.4% and 5.8%, respectively. The recoveries of amines ranged from 96 to 113%. Excellent linear responses were observed with correlation coefficients of >0.9994. The established method provided a simple and highly sensitive technique for the quantitative analysis of trace amounts of aliphatic amines from biological and natural environmental samples.