Native fluorescence detection of flavin derivatives by microchip capillary electrophoresis with laser-induced fluorescence intensified charge-coupled device detection.

To widen the scope of laser-induced fluorescence (LIF) for detection in microchip capillary electrophoresis (CE), a microchip CE LIF-ICCD (intensified charge-coupled device) system based on a tunable wavelength dye laser pumped by a pico-second pulse nitrogen laser for excitation and a spectrograph with ICCD for detection had developed to demonstrate the enhancement in detection sensitivity by the following three approaches: direct detection of native fluorescence, improvement of signal-to-noise ratio by pulse laser excitation and time delay detection, and selective spectral acquisition by multi-channel detection. Riboflavin, flavin mononucleotide (FMN) and flavin-adenine dinucleotide (FAD) have been selected as they are dietetically important and microchip CE provides a promising onsite detection method. The results indicate a strong effect of wavelength on detection sensitivity and the need to tune wavelength for direct detection. Under optimized conditions (excitation 450 nm, emission 520 nm, gate delay time 45 ns, 20 mM phosphate buffer at pH 7.1), the following results were obtained under static condition: Working ranges (0.6-350 microg/l, r > 0.99), detection limits (0.15-1.0 microg/l) and peak height repeatability (1.8-2.2% R.S.D.), all within the applicability range for body fluids or beverages such as human urine and cow milk. Baseline separation of three flavins was obtained under dynamic condition and the fluorescence spectra acquired assist the identification of alkaline-degraded products of riboflavin. Thus, the capability to check peak purity and identify unknown peaks has been demonstrated.