1980 Volume 21 Issue 5 Pages 373-380_1
A high-performance liquid-chromatographic method was developed for the determination of 5′-ribonucleotides (5′-RNT) in a wide variety of foods.
5′-RNTs such as 5′-AMP, 5′-CMP, 5′-GMP, 5′-IMP, and 5′-UMP were extracted with perchloric acid, and cleaned up by passage through an activated charcoal column. 5′-RNT in this extract was then effectively separated, identified, and determined by high-performance liquid chromatography on a macroreticular anion-exchange resin (Diaion CDR-10), utilizing the 5′-nucleotidase peak-shift reaction. 5′-RNT was completely resolved on Diaion CDR-10 with 1.5M acetic acid-ammonium acetate buffer (pH 3.4) at 60°C. The enzyme added to the extract specifically converted 5′-RNTs to the corresponding nucleosides. After the enzymic reaction, 5′-RNT peaks on the chromatogram disappeared quantitatively, and the unresolved nucleoside peaks appeared around the void volume. 5′-RNT was determined on the basis of the difference in peak height or area, including overlapped or unresolved peaks, before and after the enzymic reaction.
The average recoveries of 1.0 and 1.5mg of 5′-RNT added to 5 kinds of food (10 to 25g) were 98.9 and 99.7%, respectively. The present method was applied successfully to the determination of 5′-RNTs in many kinds of food, such as seasoning, soup, mushrooms, vegetables, fruit, fish, meat, milk, etc.
