Subjects were required to be 18�C65 years old, to be healthy, and to have smoked an average of 10 CPD or more for the past year or longer as ascertained by telephone screening. Subjects had to be self-identified non-Hispanic White or Black, OSI-744 with four grandparents of the same race. Exclusions included active medical problems; pregnancy; breast feeding; current alcohol or drug abuse; current use of smokeless tobacco, pipes, cigars, and nicotine medications; and regular use of medications other than vitamins, oral contraceptives, hormone replacements, or aspirin. Procedures Subjects were screened for eligibility by telephone. Eligible subjects were asked to come to the Clinical Research Center at San Francisco General Hospital Medical Center, where the study was explained and written consent obtained.

Questionnaires were administered regarding health history, drug use history, and smoking and tobacco dependence measures, including the Fagerstr?m Test for Nicotine Dependence (FTND; Heatherton, Kozlowski, Frecker, & Fagerstr?m, 1991). Average cigarette consumption was taken as the average number of CPD in the 3 days prior to the study visit. After completing the questionnaires, a blood sample was taken and urine collected. The time of smoking the last cigarette prior to blood and urine sampling was recorded. Plasma was assayed for concentrations of nicotine, cotinine, and trans-3 hydroxycotinine (3HC). In the early part of this study, blood was analyzed for carboxyhemoglobin. Later in the study, for technical reasons, expired-air carbon monoxide (CO) was substituted (Vitalograph Breath CO).

The urine samples were analyzed for concentrations of creatinine, nicotine and its five major metabolites, NNAL, and metabolites of several PAHs. Subjects were compensated financially for participation. The study was approved by the Institutional Review Board at the University of California San Francisco. Data from this study that focused on urine menthol in relation to biomarkers of exposure to nicotine and tobacco carcinogens have previously been published (Benowitz, Dains, Dempsey, Yu, & Jacob, 2010). Analytical Chemistry Plasma nicotine was measured by gas chromatography with nitrogen phosphorus detection using a capillary column (Jacob, Wilson, & Benowitz, 1981; Jacob, Yu, Wilson, & Benowitz, 1991). Plasma cotinine and 3HC were measured by liquid chromatography�Ctandem mass spectrometry (LC-MS/MS; Dempsey et al.

, 2004). Urine concentrations of nicotine and its metabolites cotinine, 3HC, and their respective glucuronide metabolites were measured by (LC-MS/MS), as described previously (Benowitz, Jacob, Fong, & Gupta, 1994; Dempsey et al., 2004). Urine concentrations of NNAL (free plus conjugated) and PAH metabolites, Brefeldin_A including 2-naphthol, 1,2 and 3+4 hydroxyphenanthrenes, 1-hydroxypyrene, and 2-hydroxyfluorene, were measured by LC-MS/MS (Jacob et al., 2008; Jacob, Wilson, & Benowitz, 2007).