The analysis of biological samples can provide qualitative and quantitative evidence of exposure to sulfur mustard (SM). N7-(2-hydroxyethylthioethyl) guanine (N7-HETEG) is the most abundant DNA adduct and one of the biomarkers of SM exposure. For the detection and validation of SM exposure, a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-ESI-MS/MS) method for the determination of N7-HETEG in a biomedical sample was developed, using N7-benzylguanine as the internal standard. The method was validated and showed the limit of detection of 300 pg/mL (S/N > or =10) and limit of quantitation of 850 pg/mL (S/N > or =20). The linear range was 300 pg/mL-1.28 microg/mL, with both the values of intraday and interday relative standard deviations of less than 10% (n =7) over the calibration range. The recoveries varied from 100% to 132%. The method was applied for the determination of N7-HETEG in the lung of SD rats caused by dermal exposure to SM in vivo. Under the exposure to SM of 5.5, 11, 22 and 45 mg/kg, respectively, (0.56 +/- 0.16), (0.67 +/- 0.12), (1.36 +/- 0.68) and (5.14 +/- 0.92) ng of N7-HETEG per gram of SD rat lung tissue were found after four days of exposure. The amount of N7-HETEG increased with the exposed doses, and it can act as a biomarker of sulfur mustard exposure.