Inosine, guanosine, hypoxanthine, guanine, xanthine, and uric acid were quantified by HPLC using the method described by Li et al. (2014) (link), with slight modifications. Analyses were carried out using a Zorbax SB-C18 (5 μm, 4.6 × 250 mM) column connected to an HPLC device (Agilent 1,260 Infinity Quaternary LC) with a diode array detector (Agilent Technologies, Waldbronn, Germany). Working solutions (1.3 mM) of all compounds were prepared in phosphate buffer (K3PO4, 100 mM, pH 7), cleaned and sterilized by passing the solution through a filter (0.22 μm pore size; Nalgene 176–0020 nylon), and degassed by sonication. The separation of the compounds was achieved by using an isocratic flow (0.5 mL/min) of methanol and 0.1% of acetic acid in Milli-Q water (3:97, v/v). The retention times at 245 nm were 5.00, 4.75, 2.46, 2.36, 2.24, and 2.09 min for guanosine, inosine, xanthine, hypoxanthine, guanine, and uric acid, respectively.
Compound quantification was carried out by developing standard curves built using the corresponding pure compounds (Sigma, Alcobendas, Madrid). The analyses were carried out in triplicate.
Compound quantification was carried out by developing standard curves built using the corresponding pure compounds (Sigma, Alcobendas, Madrid). The analyses were carried out in triplicate.
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