A highly specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of urinary N3-methyladenine (N3-MeA), N3-ethyladenine (N3-EtA), and N3-(2-hydroxyethyl)adenine (N3-HOEtA). Chromatographic separation was achieved on a hydrophilic interaction liquid chromatography column, with a mobile phase gradient prepared from aqueous 10 mM ammonium formate–acetonitrile (5:95 v/v, pH 4.0). Quantification of the analytes was done by multiple reaction monitoring using a triple-quadrupole mass spectrometer in positive-ionization mode. The limits of quantification were 0.13, 0.02, and 0.03 ng/mL for N3-MeA, N3-EtA, and N3-HOEtA, respectively. Intraday and interday variations (relative standard deviations) ranged from 0.6 to 1.3 % and from 3.7 to 7.5 %. The recovery ranges of N3-MeA, N3-EtA, and N3-HOEtA in urine were 80.1–97.3 %, 83.3–90.0 %, and 100.0–110.0 %, respectively. The proposed method was successfully applied to urine samples from 251 volunteers including 193 regular smokers and 58 nonsmokers. The results showed that the levels of urinary N3-MeA, N3-EtA, and N3-HOEtA in smokers were significantly higher than those in nonsmokers. Furthermore, the level of urinary N3-MeA in smokers was found to be positively correlated with the level of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (r = 0.48, P < 0.001, N = 192). This method is appropriate for routine analysis and accurate quantification of N3-MeA, N3-EtA, and N3-HOEtA. It is also a useful tool for the surveillance of alkylating agent exposure.
