Rapid analysis of trace levels of flavins by pressurized capillary electrochromatography-laser induced fluorescence detection with sulfonated N-octadecyl methacrylate monolith

Abstract

In this paper, pressurized capillary electrochromatography (pCEC) with laser induced fluorescence detection (LIF) was demonstrated as a viable approach for the separation and determination of trace flavins in human plasma, where flavins tend to be degraded ex vivo. Using a sulfonated N-octadecyl methacrylate monolithic column in isocratic pCEC separation, symmetrical peak shapes and rapid separation could be obtained in a weakly acidic mobile phase. Baseline separation of riboflavin, flavin mononucleotide and flavin adenine dinucleotide could be achieved within 4.5 min in a mobile phase containing 60% (v/v) acetonitrile and 40% (v/v) of 20 mmol L(-1) phosphate buffer (pH 4.0), with -22 kV of applied voltage and 290 psi of supplementary pressure and 0.02 mL min(-1) of flow rate. Based on a 473 nm laser diode double pumped solid state source, flavins could be determined by LIF with the detection limit (LOD) as low as 0.5 nmol L(-1) (S/N=3). The concentration ranges were 0.005-2 micromol L(-1) for RF and FMN, and 0.02-40 micromol L(-1) for FAD. Owing to the weakly acidic condition selected in this experiment, the high fluorescence quantum yields and good stability of flavins contributed to a preferable analysis. Combined with a simple clean-up procedure, this method has been proved to be effective for the rapid and selective analysis of trace levels of flavins in human plasma without sample preconcentration.