Optimization of a high-performance liquid chromatographic method for the determination of nucleosides and their catabolites. Application to cat and rabbit heart perfusates.

A high-performance liquid chromatographic method is described for the separation of nucleosides and related compounds in a single isocratic run. The separation of a standard mixture of at least thirteen compounds is achieved within 15 min on a new type of reversed-phase column 25 cm long, filled with 5-micron particles of Select B. Pre-treatment of the silica particles before silanization has given this type of reversed-phase material unique characteristics for basic and acidic compounds. Chromatograms are shown to compare the effectiveness of the Select B column for the separation of nucleosides with that of other C18 and C8 phases. The separation is achieved with 0.3 M ammonium dihydrogen phosphate (pH 4.00) and a mixture of methanol, acetonitrile and tetrahydrofuran. Detection is carried out with a variable-wavelength UV detector at 254 nm. Sample preparation and the influence of the organic solvents, pH and buffer concentration are described. To illustrate the applicability of the method, representative chromatograms are shown of perfusates of Langendorff preparations and the working hearts of cats and rabbits. Remarkable differences were obtained before and after ischaemia and before and after treatment with a nucleoside transport inhibitor. Baseline separation of cytosine, orotic acid, cytidine, uracil, uric acid, hypoxanthine, xanthine, inosine, guanosine, xanthosine, allopurinol, thymine and adenosine was achieved. The detection limit for these compounds was less than 1 ng per injection.