Direct analysis of tobacco-specific nitrosamine NNK and its metabolite NNAL in human urine by LC-MS/MS: evidence of linkage to methylated DNA lesions.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its urinary metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are the most investigated carcinogenic biomarkers of tobacco-specific nitrosamines. Here, we report the development of a sensitive and selective assay based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to simultaneously measure urinary NNK and NNAL. With the use of isotope internal standards and online solid-phase extraction, urine samples were directly analyzed without prior sample purification. The detection limits of this method were 0.13 and 0.19 pg on column for NNK and NNAL, respectively. Inter- and intra-day imprecision was <10 %. Mean recovery of NNK and NNAL in urine was 99-100 %. This method was applied to measure urinary NNK and NNAL in 101 smokers and 40 nonsmokers to assess tobacco exposure. Urinary nicotine, cotinine, N3-methyladenine (N3-MeA), and N7-methylguanine (N7-MeG) were also measured by isotope-dilution LC-MS/MS methods. The results showed that urinary NNK was not observed in all smokers. Urinary free NNAL (0.10 ± 0.09 ng/mg creatinine) and total NNAL (0.17 ± 0.14 ng/mg creatinine) were detected in all smokers. Urinary concentrations of NNAL were significantly correlated with nicotine, cotinine, N3-MeA, and N7-MeG in smokers (P < 0.001). This method enables the direct and simultaneous measurement of NNK and NNAL in urine using only 50 μL of urine. This study first demonstrated in human that urinary tobacco-specific nitrosamines metabolite (NNAL) are highly correlated with their resulting methylated DNA lesions in urine, which may help to substantiate an increased cancer risk associated with tobacco smoke exposure.
Department of Public Health, Chung Shan Medical University, Taichung, 402, Taiwan.
Chiung-Wen
CW
Hu
Yu-Wen
YW
Hsu
Jian-Lian
JL
Chen
Lai-Man
LM
Tam
Mu-Rong
MR
Chao
D000225:Adenine / Q000031:analogs & derivatives / Q000652:urine; D000328:Adult; D015415:Biomarkers / Q000652:urine; D002853:Chromatography, Liquid / Q000379:methods; D003367:Cotinine / Q000652:urine; D019175:DNA Methylation; D006147:Guanine / Q000031:analogs & derivatives / Q000652:urine; D006801:Humans; D057230:Limit of Detection; D009538:Nicotine / Q000652:urine; D009602:Nitrosamines / Q000652:urine*; D011725:Pyridines / Q000652:urine*; D012680:Sensitivity and Specificity; D012907:Smoking / Q000009:adverse effects / Q000652:urine*; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*
D015415:Biomarkers; C580784:N3-methyladenine; D009602:Nitrosamines; D011725:Pyridines; D006147:Guanine; C008450:7-methylguanine; D009538:Nicotine; C016583:4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone; C099565:4-(methylnitrosamino)-1-(3-pyridyl)-1-butan-1-ol; D000225:Adenine; D003367:Cotinine