QUANTITATION OF 1,N-6-ETHENOADENINE IN RAT URINE BY IMMUNOAFFINITY EXTRACTION COMBINED WITH LIQUID-CHROMATOGRAPHY ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

A fast, highly specific analytical method was developed to quantify 1, N-6-ethenoadenine (epsilon A) in urine of rats. epsilon A is a highly mutagenic DNA adduct generated by vinyl chloride (VC) exposures as wel l as endogenously from lipid peroxidation. epsilon A was concentrated through extraction from rat urine by immunoaffinity chromatography and quantitated by liquid chromatography/electrospray ionization mass spe ctrometry (LC/ESI-MS). The average epsilon A recovery by immunoaffinit y extraction was 66%. The LC/ESI-MS selected-ion monitoring (SIM) of t he response ratio of epsilon A to its isotopically labeled internal st andard [N-15(5)]epsilon A was linear (r(2) = 0.999) and reproducible f rom 0.15 to 30 pmol/injection. The detection limit obtained in the rou tine analysis of urine of unexposed rats was 270 fmol/sample with a si gnal-to-noise ratio (S/N) 3:1. The concentration of endogenous epsilon A was determined to be 21.6 +/- 14.8 pmol/mL (3 rats). Following port al injection of chloroethylene oxide (CEO; the putative active metabol ite of VC), the rate of epsilon A excretion in urine was greatest from 0 to 24 h, with similar to 90% of the CEO-induced epsilon A excreted. By 132 h, the excretion of epsilon A was Similar to pretreatment amou nts. The accuracy of the quantitation was 107 +/- 6% (n = 4), establis hed by analyzing urine of an unexposed rat spiked with authentic epsil on A. These data indicate that the LC/ESI-MS with immunoaffinity extra ction method is precise and accurate for epsilon A quantification. The measurement of epsilon A in urine provides a potential biomarker for exposure to chemicals and processes that form this adduct.