Abstract: A rapid and simple reversed-phase HPLC(RP-HPLC) method for the determination of adenosine and its related substances was developed. On an ODS column (5μm, 150mm×4.6mm), adenosine and its related substances (adenine, guanosine, cytidine and uridine) were separated by using 0.01 mol·L^-1 KH2PO4 (pH 3.7) -CH3OH (94:6) as mobile phase. The flow rate was 1.0 mL·min^-1, p-aminobenzoic acid was used as the internal standard and UV detection was performed at 260 um. The calibration curve showed good linearity over the range of 0.01007-0.2014 μg, r = 0.9999 and the detection limit was 0.2 ng (S/N ≥ 3). The recovery was 100.l% and the precision of the method was 0.5% (n = 8).
The method is accurate, sensitive and reproducible. It has been used for the quality control of adenosine and its preparations and also for monitoring the purification and refinement process of adenosine.
The relationship between retention behavior of the analytes and properties of mobile phase was also studied and discussed in this paper. The results can help to predict the chromatographic retention behavior and understand the retention mechanism better.

Key words: Adenosine, Adenine, Adenosine, Adenine, Guanosine, Cytidine, Guanosine, Cytidine, Uridine, HPLC, Uridine, HPLC