Capillary liquid chromatographic determination of cellular flavins

A capillary LC system was set up and optimized, in which a UV absorbance detector was used and a monolithic silica-ODS column as the separation column. Two on-line concentration techniques, namely, gradient elution mode and in-tube solid-phase ion-pair microextraction (SPIPME), were combined with the capillary LC system, which proved to be beneficial to enhance the concentration sensitivity by enabling the injection of large volumes of samples. The limits of detection at ppb levels for the flavins [riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD)] were achieved using the two techniques. For in-tube SPIPME, a monolithic silica-ODS column was employed as the extraction column, on which FAD and FMN were retained by interaction with an ion-pair reagent, tetrabutylammonium phosphate, resulting in greater than 110-fold enhancement in their concentration sensitivities relative to conventional injection method. The reproducibility and linearity of the two methods were investigated. The two methods were applied to analyze trace amounts of flavins in bacterial Escherichia coli cell extracts and recombinant flavoenzymes.