Quantitation of DNA adduct of thymidylyl(3′-5′)thymidine methyl phosphotriester by liquid chromatography/negative electrospray tandem mass spectrometry

Abstract

A rapid and selective method based on liquid chromatography/electrospray tandem mass spectrometry (LC/ESI-MS/MS) has been developed for the direct quantitation of a methyl phosphotriester DNA adduct, thymidyl (3'-5') thymidine [dTp(Me)dT] from enzymatic hydrolysates of DNA (either in vitro DNA or in cell culture) treated with MNU (N-methyl-N-nitrosourea) or MMS (methyl methane sulfonate). The lower limit of quantitation was 2 ng/mL. Linearity of the calibration curve was greater than 0.999 from 2 to 1000 ng/mL. Intraday precision for four levels of quality controls ranged from 2.8 to 20.1%, and interday precision ranged from 2.9 to 5.6%. This method was used to quantify the levels of dTp(Me)dT in enzymatic hydrolysates of DNA obtained from a series of incubations of salmon testis DNA or mouse lymphoma cells with either MNU or MMS.