Chromatographic and UV–visible spectrophotometric pKa determination of some purine antimetabolites

In this study, the chromatographic and spectrophotometric behaviors of polar 6-thioguanine (6-TGN) and 6-mercaptopurine (6-MCP) in acetonitrile (ACN)-water binary mixtures were investigated. The effect of pH of the mobile phase on the chromatographic behavior of 6-TGN and 6-MCP structurally related thiopurine analogs and determination of protonation constant/dissociation constant (sspKa) values with reversed-phase liquid chromatography (RPLC) method were determined in ACN-water binary mixtures with ACN percentages of 1, 3, 5, 7 and 10% (v/v). Spectral behavior and pKa value of these two compounds were performed by UV–Visible spectrophotometric methods in ACN-water mixtures containing 3, 5 and 7% (v/v) ACN. Chromatographic and spectrophotometric pKa data were evaluated by Microsoft Excel SOLVER and STAR programs, respectively. Aqueous pKa (wwpKa) values of 6-TGN and 6-MCP were calculated using the linear relationship between the pKa data obtained in ACN-water mixtures and macroscopic parameter values (dielectric constant and mole fraction).