Quantitation of normal and formaldehyde-modified deoxynucleosides by high-performance liquid chromatography/UV detection

A sensitive and selective method was developed for the first time to quantify simultaneously the normal and formaldehyde (FA)-modified bases in human placental DNA treated with 100 ppm FA for 20 h at 37 degrees Celsius. Digestion of DNA to deoxynucleosides with DNase I, phosphodiesterase and alkaline phosphatase occurred in that order with centrifugation steps. The normal and FA-modified deoxynucleosides were then resolved from one another and reagent blank interferences to produce selective separation through high performance liquid chromatography-ultraviolet detection at 254 nm. A C(18) reversed-phase column facilitated the resolution using 5 mm ammonium acetate and a gradient of 0-6% methanol at fl ow rates of 0.3-1.4 mL/min before column cleaning. The lower quantifiable limits for deoxyadenosine, deoxyguanosine, deoxycytidine, thymidine, N(6)-hydroxymethyldeoxyadenosine (N(6)-dA), N(2)-hydroxymethyldeoxyguanosine (N(2)-dG) and N(4)-hydroxymethyldeoxycytidine (N(4)-dC) were 11, 7.6, 12, 15, 10, 10 and 22 pmol, respectively. The abundance order of the modified deoxynucleosides was N(6)-dA > N(2)-dG > N(4)-dC. dT did not form hydroxymethyl derivatives. The respective concentrations were about 6.0, 10.0 and 23 pmol of modified deoxynucleosides in 80 micro g of human placental DNA after treatment with 100 micro g/mL of formalin for 20 h at 37 degrees Celsius. The stabilities of N(6)-dA and N(2)-dG were much better at -20 degrees Celsius than at 25 degrees Celsius, where the respective halftimes were about 50.1 and 21.0 h.