Simultaneous determination of riboflavin phosphate and other ingredients in a multivitamin pharmaceutical preparation by on-line automated LC coupled with pre-column immobilized enzyme reactor.

An automated chromatographic detection system for the simultaneous determination of riboflavin phosphate, caffeine, nicotinamide and pyridoxine hydrochloride in a multivitamin pharmaceutical preparation was constructed. Hydrolytic pretreatment of riboflavin phosphate to riboflavin was carried out using a pre-column enzyme reactor, in which immobilized sweet potato acid phosphatase was packed, and then enzymatically hydrolyzed riboflavin and other ingredients in the pharmaceutical preparation were concentrated in an ODS trap column. The concentrated riboflavin and other ingredients were back-eluted from the trap column using a mobile phase containing 1-decanesulfonate as an ion-pair reagent, and then subsequently chromatographed on an ODS analytical column. It was necessary to wash the ODS trap column with aqueous acetonitrile to remove 1-decanesulfonate in the trap column, which is advantageous to concentrate the riboflavin and other ingredients for the subsequent analysis. The calibration curves for riboflavin phosphate and other ingredients were linear over the concentration ranges tested, and correlation coefficients for standard curves were 0.9999 for all four ingredients. Analytical recoveries of the four ingredients at different levels of concentration added to the ordinary pharmaceutical preparation were also in the range of 99.1-101.2%. The present method was superior to the ordinary manual and batch-wise enzymatic methods in being harmless to the environment, rapid and accurate under continuous autoanalysis.