Automated method for analysis of tryptophan and tyrosine metabolites using capillary electrophoresis with native fluorescence detection

摘要

Capillary electrophoresis (CE) with laser-induced native fluorescence (LINF) detection offers the ability to characterize low levels of selected analyte classes, depending on the excitation and emission wavelengths used. Here a new automated CE-LINF system that provides deep ultraviolet (DUV) excitation (224 nm) and variable emission wavelength detection was evaluated for the analysis of small molecule tryptophan- and tyrosine-related metabolites. The optimized instrument design includes several features that increase throughput, lower instrument cost and maintenance, and decrease complexity when compared with earlier systems using DUV excitation. Sensitivity is enhanced by using an ellipsoid detection cell to increase the fluorescence collection efficiency. The limits of detection ranged from 4 to 30 nmol/L for serotonin and tyrosine, respectively. The system demonstrated excellent linearity over several orders of magnitude of concentration, and intraday precision from 1–11% relative standard deviation (RSD). The instrument’s performance was validated via tryptophan and serotonin characterization using tissue extracts from the mammalian brain stem, with RSDs of less than 10% for both metabolites. The flexibility and sensitivity offered by DUV laser excitation and tunable emission enables a broad range of small-volume measurements.