Comparison of UV, Fluorescence, and Electrochemical Detectors for the Analysis of Formaldehyde-Induced DNA Adducts

A high-performance liquid chromatography (HPLC) method for normal and formaldehyde-modified deoxynucleosides after hydrolysis of exposed and unexposed human placental DNA was compared with ultraviolet (UV), fluorescence (FL), and electrochemical (EC) detectors. The lower quantifiable limits (LQL) for UV detection at 254 nm were 10–22 pmol for N6-hydroxymethyldeoxyadenosine (N6-dA), N2-hydroxymethyldeoxyguanosine (N2-dG), and N4-hydroxymethyldeoxycytidine (N4-dC), with N4-dC having the highest LQL and the 2 purines the lowest LQL. Similarly, LQLs using FL (excitation: 264 nm; emission: 340 nm) were 14–30 pmol, with N2-dG having the lowest LQL and N6-dA the highest. The LQL for N2-dG by EC detection at +1.10 V was 27 pmol, over 50-fold greater sensitivity than for the other hydroxymethyl deoxynucleosides; deoxyguanosine was similarly detected more sensitively than the other normal deoxynucleosides. Percent relative standard deviations ranged between 6 and 13% for both intra- and interrun assays for all detectors. HPLC-UV allows all the deoxynucleosides to be detected without the flow cell washing and use of fresh solutions necessary for the more selective FL detection, the latter not having enzyme blank interferences. EC allows only deoxyguanosine and N2-dG to be detected at pmol levels with no blank interferences. HPLC-UV allowed more sensitive detection of N2-dG and N6-dA than the other techniques and is recommended. The UV, FL, and EC properties of the hydroxymethyl deoxynucleosides of dA, dG, and dC are reported for the first time.