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.

Sweet potato acid phosphatase immobilized on glutaraldehyde-activated aminopropyl controlled-pore glass: activation, repeated use and enzyme fatigue.

Sweet potato acid phosphatase was covalently coupled with glutaraldehyde to aminopropyl controlled-pore glass, and used as a pre-column enzyme reactor. The immobilized enzyme reactor (IMER) was continuously operated using an automated chromatographic detection system we developed. Functional evaluation of the IMER was carried out by injecting ten samples on the same day at an injection amount of 1.25 nmol (62.5 nmol per ml) using riboflavin sodium phosphate (FMNs) as a substrate, and by prolonged use for ten months. The IMER exhibited decreased activity after repeated use for a total of 3000 samples, but about 75% of its original activity remained. The conversion rate of FMNs to riboflavin by IMER was increased from 89 to 97% by adding citrate, ethylenediaminetetraacetic acid disodium salt, etc., but especially by adding citrate. The increased conversion of FMNs to riboflavin due to the addition of citrate was probably not due to the chelation of heavy metal ions by citrate. We also investigated complex formation of acid phosphatase with the substrate FMNs using surface plasmon resonance to determine the effect of citrate on the processes of association and/or dissociation between the enzyme and substrate. Enzyme fatigue was also observed during the course of prolonged and repeated use.