Development and validation of RP-HPLC method for analysis and stability study of 8-carboxyphenyl-2'- deoxyguanosine and 8-bromo-2'-deoxyguanosine

Modifications on C8 position of purine base have been studied for their effects on B-DNA to Z-DNA conversion. The 8-bromo and 8-carboxyphenyl adduct are among the most potent Z-DNA facilitators and hence are aimed to be utilized for Z-DNA based probe for binding study between Z-DNA and potential Z-DNA binding proteins. However, modification of the purine may also lead to destabilization of the nucleoside that will limit its practicality to be used for Z-DNA probe construction. In this study, the method development and validation for 2'deoxyguanosine (dG), 8-bromo-2'-deoxyguanosine (8-BrdG), and 8-carboxy-2'-deoxyguanosine (8-CpdG) determinations were performed with InertSustain® C18 (4.6 x 150 mm, 5 µm) column. A mixture of acetonitrile and ammonium acetate buffer pH 6.0 (92:8) was used as mobile phase with flow rate at 1 mL/min. A novel, simple, sensitive, precise, and accurate isocratic RP-HPLC method was obtained and used for stability evaluation of dG, 8-BrdG, and 8-CpdG. The first-order kinetic result obtained with the validated method indicate that 8-bromo and 8-carboxyphenyl adducts destabilized nucleoside unit and increased deglycosylation rate of dG, while 8-BrdG was more stable than 8-CpdG and was a suitable selection for Z-DNA probe development.