Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide in capillary zone electrophoresis with laser-induced fluorescence detection

Abstract

A method was developed for the quantitative determination of riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), using free solution capillary zone electrophoresis in uncoated fused-silica capillaries with laser-induced fluorescence (LIF) detection. Various factors influencing the separation and detection of flavin vitamers were investigated, including pH (5.5–10.5), concentration and nature of the run buffer (phosphate, borate and carbonate), applied voltage (15–30 kV), temperature (15–30 °C) and injection time. Optimal resolution and detection were obtained with a pH 9.8, 30 m M aqueous phosphate buffer at 15 °C and 30 kV of applied voltage. LIF detection was obtained with a He–Cd laser source using an excitation wavelength at 442 nm and λ em≥515 nm. Riboflavin could be determined in the concentration ranges 0.5–350 μg/l with a rather low detection limit (LOD) down to 50 amol. The LODs of FAD and FMN were slightly higher, 300 and 350 amol, respectively. Combined with a simple clean-up procedure, the practical utility of this method is illustrated by the measurements of flavin derivates in foods and beverages, such as wines, milk, yoghurt and raw eggs.