Abstract
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N-and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter-individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre-ionized N-propyl-NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on-going longitudinal biomarker studies. The new high sensitivity SID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease-free smokers. Effects of inter-individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1). Copyright (C) 2010 John Wiley & Sons, Ltd.