Development of an analytical method for the determination of ingredients in multivitamin pharmaceutical preparations using immobilized enzyme reactors

Automated detection systems using immobilized enzyme reactors for the determination of riboflavin phosphate (FMNs) and myo- inositol (Ml) in a multivitamin pharmaceutical preparation were constructed. A high-performance liquid chromatographic detection system using a pre-column enzyme reactor, in which immobilized sweet potato acid phosphatase was packed, was developed for the simultaneous determination of FMNs and other water-soluble vitamins. 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, and the chromatographed on an ODS analytical column. The effluents were monitored by UV absorption at 280 nm. An automated flow-injection system using an enzyme reactor, in which immobilized myoinositol dehydrogenase was packed, was developed for the specific determination of MI. MI was detected using a fluorophotometer, to measure the fluorescence of NADH produced from NAD+ by the reactor. Enhanced interference due to excess glucose included in a pharmaceutical preparation as a sweetener was eliminated by a glucose eliminating reactor co-immobilized with three enzymes (glucose oxidase, mutarotase and catalase). The two automated detection system for the determination of ingredients in pharmaceutical preparations was superior to ordinary manual methods in terms of being harmless to the environment, rapidity and accuracy under continuous autoanalysis.