Profiling of amine metabolites in human biofluids by micellar electrokinetic chromatography with laser-induced fluorescence detection

Abstract

A rapid and sensitive capillary electrophoresis (CE) method has been developed for profiling organic metabolites containing amine functional groups in mammalian biofluids. Metabolites containing an amine group were derivatized with 4-fluoro-7-nitrobenzo-2,1,3-oxadiazol (NBD-F), separated by micellar electrokinetic chromatography (MEKC), and detected by argon-ion (488 nm) laser-induced fluorescence detection. The optimized MEKC background electrolyte conditions were: 50 mmol L⁻¹ sodium cholate, 5 mmol L⁻¹ β-cyclodextrin, and 20 mmol L⁻¹ Brij 35 in 20 mmol L⁻¹ aqueous borate buffer, pH 9.3, containing 7% methanol. Under these conditions all the amine compounds in mammalian biofluids, for example plasma, saliva, and urine, were derivatized directly, without extraction, in a minimum volume of 100 nL and the derivatives could be separated within 16 min. Up to 90% of the amine-containing metabolites in plasma and saliva could be identified by reference to standard compounds. For twenty amine standards linearity of calibration was better than R ² = 0.99. Migration-time and peak-area reproducibility were better than RSD 1.5% and 15% respectively. In replicate analysis of human plasma bioanalytical precision ranged between 0.7 and 3.8 RSD% for a 5.0-μL volume and between 1.7 and 5.5 RSD% for 100-nL volume. The concentrations measured were found to be in agreement with literature values.