Optimization of High Performance Liquid Chromatography Method for Simultaneous Determination of Some Purine and Pyrimidine Bases.

Nine purine and pyrimidine bases were separated and determined simultaneously using reversed phase (RP) high performance liquid chromatography (HPLC) in some food samples and biological fluids. Chromatographic behavior of these ionizable compounds highly depends on the interactions with the solvent as confirmed experimentally and by calculation of distribution of this species as a function of pH. Chromatograms show the optimal separation of five purine (uric acid, hypoxanthine, xanthine, adenine, and guanosine), and four pyrimidine (cytosine, uracil, cytidine and tymine) bases at pH around four. Accordingly, acetate buffer was selected due to high buffer capacity in this region. By variation of pH, concentration of buffer and volume ratio between buffer and methanol, we found that a mixture of 50 mM acetate buffer of pH 4.0 ± 0.1 with 3 % of methanol ensures reproducibility, complete separation in less than 15 minutes and compatibility with MS detection. Developed method was validated and applied for the analysis of complex clinical and beverage samples.