Quantitation of 5′-nucleotides in enzymatic hydrolysis products of ribonucleic acid for injection by reversed phase ion-pair high performance liquid chromatography

Objective To establish a quantitative method by reversed phase ion-pair HPLC to simultaneously determine 4 types of 5′-nucleotides in enzymatic hydrolysis products in ribonucleic acid for injection.The concentrations of the 5′-nucleotides were summed to obtain the original concentration of ribonucleic acid in the injections.Methods The sample was hydrolyzed by snake venom phosphodiesterase(SVP) to generate 5′-nucleotides.The separation was achieved on a SHIMADZU VP-ODS column(250 mm×4.6 mm,5 μm) proceded with a C18reversed phase cartridge guard column with the mobile phase consisting of buffer A [20 mmol L-1 KH2PO4 buffer,1% tetrabutylammonium hydroxide(TBAOH)(10% aqueous solution),adjusted to pH = 6.5 and 5% methanol] and buffer B [1% TBAOH(10% aqueous solution),adjusted to pH = 6.5 and 80% methanol] with a gradient elution.Chromatogram was obtained at 254 nm.Results All the 5′-nucleotides were well separated when an optimized elution condition was applied.The calibration curves of 5′-nucleotides showed good linearity at 0.50-15 μg mL-1,and the correlation coefficient was 0.999 8,0.999 9,0.999 9,and 0.999 9,respectively.The average recovery was 95.1%-106.3% with RSD 3.5%(n = 6).Conclusion With good reproducibility,specificity and precision,this method can be used for the determination of nucleotides hydrolyzed in ribonucleic acid for injection.