Quantitative analysis of etheno-2 '-deoxycytidine DNA adducts using on-line immunoaffinity chromatography coupled with LC/ES-MS/MS detection

Etheno DNA adducts, including 3,N-4-etheno-2'-deoxycytidine (etheno-dC), ar e promutagenic lesions present in normal animal and human tissues. These DN A adducts are believed to be important in the etiology of cancer. Existing methods for quantifying etheno-dC use P-32. postlabeling, Although highly s ensitive, postlabeling requires the use of an energetic radioisotope and co nsiderable time and effort. The new methodology reported here permits autom ated quantitication of trace levels of etheno-dC in crude DNA hydrolysates on the order of 5 adducts in 10(8) normal nucleotides from 100-mug samples of DNA. This was accomplished by using on-line immunoaffinity chromatograph y, a reverse-phase LC separation on graphitized carbon, tandem mass spectro metric detection, and an isotopically labeled internal standard. The automa ted procedures permitted analysis of 4 DNA hydrolysates/hr. The sensitivity using immunoaffinity cleanup was approximately 100-fold greater than that observed when using a silica-based trapping system. The validated method wa s applied to the analysis of etheno-dC in commercial calf thymus DNA, untre ated mouse liver, and untreated rat liver DNA, The demonstrated level of pe rformance suggests future applicability of this method in studies of cancer in humans and experimental animals.