title: New validated LC-MS/MS method for the determination of three alkylated adenines in human urine and its application to the monitoring of alkylating agents in cigarette smoke.
abstract: A highly specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for simultaneous determination of urinary N(3)-methyladenine (N(3)-MeA), N(3)-ethyladenine (N(3)-EtA), and N(3)-(2-hydroxyethyl)adenine (N(3)-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 N(3)-MeA, N(3)-EtA, and N(3)-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 N(3)-MeA, N(3)-EtA, and N(3)-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 N(3)-MeA, N(3)-EtA, and N(3)-HOEtA in smokers were significantly higher than those in nonsmokers. Furthermore, the level of urinary N(3)-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 N(3)-MeA, N(3)-EtA, and N(3)-HOEtA. It is also a useful tool for the surveillance of alkylating agent exposure.
authors.affiliation: China National Tobacco Quality Supervision and Test Center, No.2, Zhengzhou, 450001, Henan, China.
authors.forename: Yongfeng
authors.initials: Y
authors.lastname: Tian
authors.forename: Hongwei
authors.initials: H
authors.lastname: Hou
authors.forename: Xiaotao
authors.initials: X
authors.lastname: Zhang
authors.forename: An
authors.initials: A
authors.lastname: Wang
authors.forename: Yong
authors.initials: Y
authors.lastname: Liu
authors.forename: Qingyuan
authors.initials: Q
authors.lastname: Hu
mesh_terms: D000225:Adenine / Q000652:urine*; D000477:Alkylating Agents / Q000032:analysis*; D015415:Biomarkers / Q000032:analysis / Q000652:urine; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D018736:DNA Adducts; D019175:DNA Methylation; D006801:Humans; D015203:Reproducibility of Results; D012906:Smoke / Q000032:analysis*; D012907:Smoking / Q000652:urine*; D053719:Tandem Mass Spectrometry / Q000379:methods*; D062789:Tobacco Products
chemical_list: D000477:Alkylating Agents; D015415:Biomarkers; D018736:DNA Adducts; D012906:Smoke; D000225:Adenine