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 37degreesC. Digestion of DNA to deoxynucleosides with DNase 1, 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 flow 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-hydroxymethyideoxycytidine (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 mug of human placental DNA after treatment with 100 mug/mL of formalin for 20 h at 37degreesC. The stabilities of N-6-dA and N-2-dG were much better at -20degreesC than at 25degreesC, where the respective halftimes were about 50.1 and 21.0 h. Copyright (C) 2004 John Wiley Sons, Ltd.