On-line automated high-performance liquid chromatographic determination of total riboflavin phosphates using immobilized acid phosphatase as a pre-column reactor

An automated chromatographic detection system for the determination of total riboflavin phosphates using immobilized sweet potato acid phosphatase as a pre-column reactor is reported on. An immobilized enzyme reactor, incorporated in the on-line analytical system, hydrolysed riboflavin phosphates to riboflavin, and then lipophilic riboflavin was concentrated at the top of an ODS trap column. Enzymatically hydrolysed riboflavin was back-eluted from the trap column using a mobile phase containing methanol, and then subsequently chromatographed on an ODS analytical column. The effluents were monitored by UV absorption at 280 nm. The calibration graph for total riboflavin phosphates, determined by this method, was linear over the range 0.5–500 nmol/ml, with a correlation coefficient of 0.9999. The detection limit at a signal-to-noise ratio of 3 was 25 pmol/ml. The average conversion rate of riboflavin phosphates to riboflavin was estimated at 97%. The relative standard deviations of the intra- and inter-assay precision were 1.2 and 2.6%, respectively.