Abstract
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.
Highlights
Purine and pyrimidne bases and their nucleotides are involved in a numerous biochemical processes, and they play an important role in cell metabolism as monomeric precursors of RNA and DNA and as a secondary messenger.[1]
Since the majority of previous reports on high performance liquid chromatography (HPLC) separation of purine and pyrimidine bases were performed with phosphate buffers[2,5,6,9,10,11,12,15] which according to our observation do not ensure satisfactory separation and reproducible retention in acidic region, the main aim of our research was to find better mobile phase and to optimize the conditions for their complete separation
Purine and pyrimidine bases are ionizable compounds and concentration of their species highly depends on the pH of the mobile phase
Summary
Purine and pyrimidne bases and their nucleotides are involved in a numerous biochemical processes, and they play an important role in cell metabolism as monomeric precursors of RNA and DNA and as a secondary messenger.[1] Their determination is very important in many areas of research, like clinical analysis,[2,3,4,5,6,7] or food and beverage analysis. Many analytical methods have been developed for determination of purine and pyrimidine bases. The most widely used technique for biological samples is reversed phase (RP) or RP ion-pairing[15] high performance liquid chromatography (HPLC) because it gives reproducible results, and is sensitive, selective and automated.[2,3,5,6,7,10,12,13,14] Capillary electrophoresis is widely used for the separation of these analytes because of its minimal sample volume, short analysis time and high separation efficiency.[16,17,18] For both techniques UV/VIS,[5,18] electrochemical[19] or MS detection is used.[13,14,18,20,21,22] Since nucleobases are electroactive, in some papers voltammetric determination with static mercury drop electrode, by modified graphite/carbon electrodes,[23,24] and on the boron-doped carbon nanotubes are reported.[27]
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