To access total nicotine, and hence tobacco smoke exposure, the urinary concentration of the sum of nicotine and 5 nicotine metabolites was determined. The sum of these 6 compounds, which account for 75−95% of the nicotine dose is referred to as nicotine equivalents and is considered the most comprehensive measure of exposure to nicotine (11). Specifically we measured the molar concentrations of total nicotine (free plus nicotine N-glucuronide), total cotinine (free plus cotinine N-glucuronide), total trans-3′-hydroxycotinine (3-HC) (free plus 3-HC N- and O-glucuronide), as well as a metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and its detoxification product NNAL-glucuronide (NNAL-Gluc), in the 12-hour urine. Nicotine metabolites were measured by gas chromatography-mass spectrometry (GC-MS), and nicotine equivalents calculated as the sum of the molarity of total nicotine, total cotinine and total 3-HC. NNK metabolites were measured by GC with nitrosamine selective detection (GC-TEA) (9,10). Based on 65 blind duplicate pairs analyzed with the study samples for total nicotine and total cotinine, and 6 pairs for total 3HC, the intraclass correlation coefficient was 0.98, 0.96 and 0.62, respectively. NNK metabolites were determined as described with slight modifications (12).
