Quantitation of N'-Nitrosonornicotine (NNN) in Smokers Urine by Liquid Chromatography Tandem Mass Spectrometry

Transcription

1 Quantitation of N'-Nitrosonornicotine (NNN) in Smokers Urine by Liquid Chromatography Tandem Mass Spectrometry Michael Urban 1, Gerhard Scherer 1, *, Dominique Kavvadias 1, Heinz-Werner Hagedorn 1, Shixia Feng 2, Richard Serafin 2, Sunil Kapur 2, Raheema Muhammad 2, Yan Jin 2, Paul Mendes 2, and Hans Roethig 2 1 Analytisch-Biologisches Forschungslabor GmbH, Goethestrasse 20, München, Germany and 2 Altria Client Services, Research Development & Engineering, 601 E. Jackson Street, Richmond, Virginia Abstract The tobacco-specific nitrosamine N'-nitrosonornicotine (NNN) is carcinogenic to humans (IARC Group 1). Assessing the tobacco smoke-related exposure to NNN by suitable biomarkers is of interest for risk evaluation. Recently, NNN and NNN-Nglucuronide have been quantified in urine of smokers. However, it is unknown what percentage of the absorbed dose of NNN is excreted as total NNN (sum of free and conjugated NNN) in urine of smokers. We developed a sensitive method based on liquid chromatography with tandem mass spectrometry with deuterium-labeled internal standard for the determination of total NNN in human urine. The limit of quantitation of the method was 2 pg/ml with a calibration line linear up to 256 pg/ml. In a study with 16 smokers in which the respiratory retention of NNN was measured through controlled smoking, we found that on average about 1% of the pulmonary NNN dose was excreted in 24 h urine as total NNN. Introduction N'-Nitrosonornicotine (NNN) is a tobacco-specific N-nitrosamine (TSNA), which is mainly formed from nornicotine during the curing process of tobacco leaves (1 3). TSNA levels in tobacco vary widely and are significantly correlated with the amount of nitrate in tobacco (4). Median yields of NNN in mainstream smoke of cigarettes when smoked according to the Massachusetts intense smoking regime were found to be ( ) ng/cigarette (5). The International Agency for Research on Cancer (IARC) has classified NNN as a Group 1 carcinogen (carcinogenic to humans) (6). Based on the experience with laboratory animals, NNN is believed to play a role in causing esophageal cancer in smokers (7) and oral cancer in smokeless tobacco users (6). NNN metabolism in vivo has been extensively investigated in * Author to whom correspondence should be addressed. Gerhard.Scherer@abf-lab.com. laboratory animals (8). A pharmacokinetic study in patas monkeys revealed norcotinine, norcotinine-1-n-oxide, 3'-hydroxynorcotinine and its O-glucuronide as well as keto acid, and hydroxy acid as metabolites in urine (9). The bulk amount of these metabolites are unspecific and could also form from other compounds such as nicotine, nornicotine, or cotinine. Unchanged NNN accounted for 0.63% of all urinary NNN metabolites (9). After α-hydroxylation at the 2'-position, NNN forms the same 4-hydroxy-1-(3-pyridyl)-butanone (HPB) releasing adducts with proteins and DNA as does 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, another carcinogenic TSNA). However, NNN forms these adducts to a significantly lower extent than NNK (8). Recently, NNN-N-glucuronide, along with NNN, was identified in urine of smokers and smokeless tobacco users (10). The structures of NNN and NNN-N-glucuronide are shown in Figure 1. Using a gas chromatograph with a nitrosaminespecific thermal energy analyzer (GC TEA) method which had a detection limit of pmol/ml (equivalent to 5.7 pg/ml), NNN was detected in 10 of 14 smokers urine samples and 11 of 11 smokeless tobacco users urine samples (10). The authors reported a good correlation between urinary total NNN excretion and urinary total cotinine or total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, the Figure 1. Structures of NNN and NNN-N-glucuronide. 260 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher s permission.

2 major metabolite of NNK). It was suggested that the sum of free NNN and NNN-N-glucuronide (total NNN) in urine could serve as a specific urinary biomarker of human NNN uptake. However, the relationship between urinary NNN levels and the NNN dose from smoking is unknown in humans. In this report, we describe the development of a more sensitive liquid chromatography tandem mass spectrometry (LC MS MS) method for total NNN in human urine in order to enhance the ability to monitor human exposure to NNN. The method was applied to urine samples from a controlled clinical study in adult smokers, during which NNN exposure was estimated from the retained NNN amount. Experimental Standards and supplies NNN (purity 98%) was purchased from Toronto Research Chemicals (North York, ON, Canada) and pyridine-ringdeuterated N'-nitrosonornicotine (NNN-d 4 ) was obtained from CDN Isotopes (Québec, QC, Canada). Disodiumhydrogen phosphate ( 99.5 %) and β-glucuronidase (type IX-A, E. coli) were obtained from Sigma-Aldrich (Steinheim, Germany). Potassium chloride and hydrochloric acid (> 37%), were supplied by Riedel-de Haën (Seelze, Germany). Potassium dihydrogen phosphate was purchased from Merck (Darmstadt, Germany). Methylene chloride, hexane, methanol (all nitrosamine free), ethyl acetate, toluene ( 99.9%), and acetonitrile were purchased from Promochem GmbH (Wesel, Germany). All other chemicals were of analytical grade or better and procured from Sigma-Aldrich. Urine sample preparation for the determination of total NNN Twenty milliliters of urine was adjusted to ph 7.2 with 1 N sodium hydroxide (NaOH) followed by addition of 2 ml 0.1 M phosphate buffer (ph 7.2). The ph was measured by means of a ph-electrode. The mixture was incubated with 5000 units β-glucuronidase (Type IX-A from E. coli) at 37 C for 16 h. After re-adjusting the ph to 7.2 with 1 N NaOH, 4 ng NNN-d 4 in 40 µl methanol was added as internal standard. The hydrolyzed sample was applied to Extrelut NT 20 cartridges (Merck, Darmstadt, Germany). The Extrelut cartridges contained 17.2 g of inert diatomaceous earth (bed dimensions: 10-cm length, 2.7-cm diameter) suitable for absorbing about 20 ml aqueous sample. After 30 min, the cartridge was eluted with 42 ml methylene chloride/isopropanol (9:1, v/v). The eluate was evaporated to dryness by means of a Tubovap evaporator (Zymark, Idstein, Germany) and the residue dissolved in 10 ml methylene chloride. The extract was liquid liquid extracted three times with 5 ml of 0.4 M hydrochloric acid. The combined extracts were subjected to solid-phase extraction on a cation exchange cartridge (Oasis MCX 1 g, 20 ml, Waters GmbH, Darmstadt, Germany) that was conditioned with 40 ml methanol, 40 ml phosphate buffered saline (PBS), and 12 ml 0.4 N hydrochloric acid. The loaded cartridge was washed with 8 ml methanol, 8 ml acetonitrile, and 8 ml toluene. The cartridge was dried in a stream of nitrogen and eluted with 8 ml ethyl acetate/20% methanol in ethyl acetate saturated with ammonia. The eluent was evaporated to dryness (Speed Vac evaporator), the residue was re-dissolved in 300 µl ethyl acetate/20% methanol (saturated with ammonia), transferred to microvials, evaporated to dryness again, and re-dissolved in 50 µl mobile phase used at start of chromatography. Analysis of total NNN by LC MS MS Ten microliters of the final extract was injected into an LC MS MS system (Sciex API 4000, Applied Biosystems, Darmstadt, Germany) equipped with two analytical columns connected in series (Luna Aqua C A, mm, 5-µm particle size and Synergi Polar-RP 80A silica, mm, 4-µm particle size, Phenomenex, Aschaffenburg, Germany). Chromatography was performed isothermally at 50 C with a gradient consisting of 15 mm ammonium acetate (A) and 0.1% formic acid in acetonitrile (B) at 0.9 ml/min flow rate. The following gradient was used: 0.00 min: 70% A, 1.00 min: 70% A, 5.00 min: 5% A, 9.00 min: 5% A, 9.01 min: 70% A, min: 70% A. Retention time was 8.17 for NNN and NNN-d 4. Positive electrospray ionization (ESI + ) was applied, and the MS MS was run in the multiple reaction monitoring (MRM) mode using the following mass transitions: NNN: m/z (quantifier), m/z (qualifier), NNN-d 4 : m/z (quantifier), m/z (qualifier). For calibration, a nonsmoker pooled urine was spiked at the following concentrations of NNN: 2, 4, 8, 16, 32, 64, 128, and 256 pg/ml. Linear regression was applied for calculating the calibration function. A quality control sample (smoker pool urine at 12.4 pg/ml NNN) was run every 16th sample. A tolerance of ±15% from the pre-determined target value was accepted. Validation of the analytical method for total urinary NNN Intraday precision was determined by measuring total NNN at three levels (low, medium, and high) in pooled urine samples 9 10 times each. The low and medium level samples were mixtures of nonsmokers and smokers urine pools. The high level sample was a smoker urine pool fortified with NNN. Interday precision was determined by measuring total NNN in the low, medium, and high level pooled urine samples on 8 11 different days. Accuracy was determined by spiking a nonsmoker pool urine with 2.0, 6.6, and 11.6 pg/ml NNN. Each level was measured 4 6 times. The recovery of the analytical procedure was determined by comparing the peak area of the internal standard (NNN-d 4 ) added at the beginning of the clean-up procedure (usual procedure as described) with the peak area obtained when adding the internal standard to the eluate of the solid-phase extraction on cation exchange resin, right before injection into the LC MS MS system. The limits of detection (LOD) and quantification (LOQ) were determined with the signal/noise method using the integrated function of the LC MS MS system (Analyst 1.4.2, Applied Biosystems). Signal/noise ratios of 3 and 10 were applied for estimating the LOD and LOQ, respectively. 261

3 The dependency of total NNN yields on the amount of β-glucuronidase was determined by hydrolyzing a smoker urine sample with 625, 1250, 2500, 5000, 6660, 10,000, and 20,000 units of β-glucuronidase from E. coli per 20 ml urine (37 C, 16 h). Possible artifactual formation of NNN during the analytical procedure and during storage of freshly colleted urine was investigated. Freshly collected nonsmoker urine (ph 6.41) was aliquoted into 20-mL samples, which were treated as follows: Aliquot 1: stabilized by addition of ~ 80 µg NaOH, stored for 2 h at 21 C (blank control); Aliquot 2: addition of ~ 80 µg NaOH, 10 µg nornicotine, 200 µg nitrite, 2000 µg nitrate, stored for 2 h at 21 C; Aliquot 3: addition of 10 µg nornicotine, 200 µg nitrite, 2000 µg nitrate, stored for 2 h at 21 C; and Aliquot 4: addition of 10 µg nornicotine, 200 µg nitrite, 2000 µg nitrate, stored for 19 h at 21 C. All aliquots were analyzed for total NNN. Clinical study in adult smokers Urine samples were collected from a previously reported controlled clinical smoking study (11,12). The study was approved by an Institutional Review Board and all subjects gave written consent for participating in the study. Sixteen healthy adult male smokers (21 29 years old) stayed in a clinic for eight days and received a controlled diet. After a four-day washout period (Day 4 to Day 1), each subject smoked six cigarettes (a commercial brand with 11-mg tar delivery according to the Federal Trade Commission testing method) per day for four consecutive days (Day 1 to Day 4) according to three predefined smoking patterns: no inhalation (Pattern A), normal inhalation (Pattern B), and deep inhalation (Pattern C). The amount of NNN retained in the respiratory tract during smoking was estimated from the difference between the amount of NNN inhaled and exhaled (12). All urine voided by each subject over a 24-h period (7:00 a.m. to 7:00 a.m.) from the first day through the end of the study was collected and kept frozen at 20 C until analysis (18 months). NNN in urine was found to be stable under these conditions for at least 18 months. Data analysis SAS for Windows (Cary, NC, release 9.1) was used for conducting the statistical analyses. The estimated total daily NNN dose recovered as total NNN in urine was calculated as Recovery (%) = 100 (amount excreted/total daily uptake) where amount excreted = NNN concentration urine volume. Descriptive statistics were calculated for total daily uptake of NNN, daily NNN excretion in urine, and daily NNN dose recovered. Results Figure 2. Product ion spectra of NNN (A) and NNN-d 4 (B) with suggested chemical structures of ion fragments Analytical method for total urinary NNN Product ion spectra for NNN and the internal standard NNNd 4 are shown in Figure 2. Typical chromatograms of the mass transitions of NNN (m/z ) and its deuterated internal standard NNN-d 4 (m/z ) for a urine sample of a smoker are shown in Figure 3. The amount of total NNN determined in a smoker urine sample increased with the amount β-glucuronidase added for enzymatic hydrolysis of the NNN-N-glucuronide, up to an amount of 5000 units per 20 ml, where a plateau was reached. This amount of enzyme was, therefore, used for all further analyses. The method performance data are summarized in Table I. Intra- and interday precisions were found to be well below 15% for total NNN in urine. Observed accuracies were in the range of %. Extraction recovery rates for NNN-d 4 were found to be 79.7% and 67.6% for smoker and nonsmoker urine samples, respectively. LOD and LOQ of NNN in urine matrix were found to be 0.4 and 1.3 pg/ml, respectively. The lowest calibrator used was 2 pg/ml. No additional formation of NNN was observed when nornicotine, nitrite, and nitrate were added to the urine sample immediately before start of the sample clean-up procedure. No NNN was found in the NaOH-stabilized samples (Aliquots 1 and 2 as described in the Experimental section). Concentra- 262

4 tions of 2 and 7.5 pg/ml were found in Aliquots 3 and 4, respectively, which were stored without stabilization by NaOH. Clinical study Table II shows the total daily amount of NNN retained (pulmonary dose) from smoking six study cigarettes in relation to the 24 h urinary excretion of NNN. The mean daily pulmonary NNN dose retained over the 4 days was 669 (ng/day) ranging from 660 to 674 (ng/day). Total urinary NNN excretions were 5.2, 6.3, 7.3, and 6.6 (ng/day) on Days 1, 2, 3, and 4, respectively. The recovered NNN dose as total urinary NNN amounted to 0.8, 1.0, 1.1, and 1.0% on Days 1, 2, 3, and 4, respectively, with a mean over the four days of 1.0%. Discussion Analytical method The newly developed LC MS MS method for the determination of NNN in urine of smokers comprises the following steps: 1. enzymatic hydrolysis of 20 ml urine with β-glucuronidase; 2. sorbent-based liquid liquid extraction, (S)LLE on diatomaceous earth (Extrelut) after addition of NNN-d 4 as internal standard; 3. liquid liquid back-extraction into acid; 4. solid-phase extraction on a cation-exchange resign; and 5. LC MS MS analysis with positive electrospray ionization and quantification in the MRM mode. The ion fragments for NNN and NNN-d 4 (Figure 2) were similar to those reported by others (13). The major fragment ions [MH 30] +, which are derived from loss of NO, were used for quantification. The method performance (Table I) shows that application to clinical or epidemiological studies is possible. Precision (both intra- and interday) was well below 15%. Accuracies ranged from to 112.5%. The extraction recovery for NNN over the whole analytical procedure was approximately 68 80%, which is acceptable, particularly because any variations in the extraction recovery rate from sample to sample are controlled by the use of the deuterated internal standard. LOD and LOQ for urinary NNN were 0.4 and 1.3 pg/ml, respectively, allowing quantification of NNN in all subjects when smoking. The results of our experiments on artifactual NNN formation suggest that the analyte is not formed during the analytical procedure, when the precursors nornicotine, nitrite, and nitrate were present in excessive amounts. The same is true for urine samples which were stored below 20 C. However, storage at room temperature for only 2 h in the presence of the precursors was found to lead to measurable NNN formation, which increased over time. As shown previously (14,15), addition of a strong base to the sample prevents Table I. Performance Data for the Method of Total NNN in Urine Parameter Data Obtained Precision Intraday 3.4 pg/ml: 11.3% (N = 9) 13.3 pg/ml: 4.7% (N = 9) 47.3 pg/ml: 1.2% (N = 10) Interday 3.4 pg/ml: 10.4% (11 days) 13.3 pg/ml: 6.1% (11 days) 47.3 pg/ml: 2.6% (8 days) Accuracy Level 1 (2.0 pg/ml, N = 2) ( ) % Level 2 (6.6 pg/ml, N = 6) ( ) % Level 3 (11.6 pg/ml, N = 6) ( ) % Extraction recovery (whole analytical procedure) Nonsmoker urine 67.6% Smoker urine 79.7% Figure 3. Chromatograms showing the mass transitions of NNN m/z (4.2 pg/ml) (A) and the added internal standard NNN-d 4 m/z in a smoker s urine (B) and m/z in a nonsmoker s urine (C). LOD (S/N = 3) LOQ (S/N = 10) Linearity and calibration function Stability in urine during storage 0.4 pg/ml 1.3 pg/ml Calibration line 2 pg/ml pg/ml y = x R 2 = Stable at 20 C for at least 18 months 263

5 artifactual formation of NNN. In order to avoid artifactual formation of NNN, it appears, therefore, advisable to store the urine at cool temperatures or to stabilize it by addition of a strong base. Stepanov and Hecht (10) were the first to determine NNN and its pyridine-n-glucuronide in urine of smokers and smokeless tobacco users. Their method for the determination of total NNN comprised enzymatic hydrolysis of the N-glucuronides, two liquid liquid extractions, two solid-phase extractions, and analysis with GC TEA. 5-Methyl-N'-nitrosonornicotine was added as internal standard after the enzymatic hydrolysis. GC TEA peak identity was confirmed in some urine samples by GC MS MS using two mass transitions for NNN. The reported method performance was to a large extent similar to that of our method. Extraction recovery for NNN in urine was 79%, which is at the upper range of the recovery of our method (68 80%). The reported LOD for NNN was 5.7 pg/ml, which is higher than in our method (0.4 pg/ml). Stepanov and Hecht (10) did not report artifactual formation of NNN during the analytical procedure. The same authors recently reported (16) an LC MS MS method for NNN in human toenails using [ 13 C 6 ]NNN as an internal standard. The toenail sample was digested with NaOH but did not involve enzymatic hydrolysis. The authors did not report the detailed method performance data. Total urinary NNN excretion in smokers Total NNN was detectable (> 0.4 pg/ml) in all 128 urine samples of the controlled smoking study. During the washout period (Day 4 to Day 1), when subjects did not smoke, NNN in all samples was below the LOQ of 2.0 pg/ml. On Days 1 4, when smoking was allowed, almost all samples (> 95%) were quantifiable for total NNN (> 2.0 pg/ml). After smoking was resumed, NNN levels reached the average smoking level of about 4 pg/ml (6 7 ng/day) within 1 day. The average total NNN concentration in our study was more than 10-fold lower compared to that reported by Stepanov and Hecht (10) (4 vs. 47 Table II. Results of the Retention Study (mean ± standard deviation, range)* Total NNN Total NNN NNN Dose Recovery NNN Dose in Urine in Urine as Total NNN Day (ng/day) (ng/day) (pg/ml) in Urine(%) ± ± ± ± 0.3 ( ) ( ) ( ) ( ) pg/ml). Several reasons might have contributed to this difference: 1. difference in the number of cigarettes; 2. difference in NNN mainstream delivery due to different types of cigarettes; and 3. methodological differences such as specificity of the detection, control of extraction recovery by the internal standard or incomplete enzymatic hydrolysis of NNN-Nglucuronide. The Stepanov and Hecht (10) study did not report the number of cigarettes or type of cigarettes smoked by the study subjects. As to the latter issue, we used the same type of β-glucuronidase (type IX-A, E. coli), but in lower amounts (250 vs U/mL) than Stepanov and Hecht (10). A systematic investigation of the dependency of yield of free NNN on the amount of β-glucuronidase used for hydrolysis showed that a plateau was reached at 250 U/mL, the amount of enzyme used in our study. We have previously reported (12) the amounts of NNN inhaled and exhaled during smoking each individual cigarette. Thus, the dose of NNN from smoking each cigarette can be calculated as the difference between the amount of NNN exhaled and inhaled. The average NNN daily dose for the 16 smokers on 4 consecutive days was ng/day (mean: 669 ng/day). The daily amount of total NNN excreted into the urine reflected an average of 1% of the absorbed NNN dose (Table II). To our knowledge, this is the first study which estimated the percentage of the inhaled dose of NNN excreted as total NNN in urine of cigarette smokers. It is interesting to note that patas monkeys, administered tritiated NNN intravenously were found to excrete 0.63% of all urinary NNN metabolites as unchanged, free NNN (9). Results from Stepanov and Hecht (10) and from our study (data not shown) suggest that free NNN represents about 50% of the total NNN excreted in urine. Assuming a similar ratio of free and glucuronidated NNN in patas monkeys would mean that about 1.2% of the absorbed NNN is excreted as total NNN in urine. This percentage is very close to that observed in our study with cigarette smokers. Because the major NNN metabolites are unspecific and could also form from other compounds such as nicotine, the unchanged NNN and its glucuronide can be measured as a specific biomarker for NNN exposure using a sensitive analytical method as described here. In addition, the method should be potentially useful to study smokeless tobacco products users ± ± ± ± 0.5 ( ) ( ) ( ) ( ) ± ± ± ± 0.4 ( ) ( ) ( ) ( ) ± ± ± ± 0.3 ( ) ( ) ( ) ( ) Mean ± ± ± ± 0.4 (Days 1 4) ( ) ( ) ( ) ( ) * Urinary NNN levels are expressed as daily excretion (ng/day) and as concentration (pg/ml). Conclusions A sensitive and specific analytical method for the determination of total NNN in human urine was developed. The method performance data meet the commonly accepted criteria for bioanalytical methods. Comparison between the NNN dose from smoking and urinary NNN excretion showed that about 1% of the NNN dose retained is excreted as total NNN in urine. 264

6 Acknowledgments The authors thank Drs. Kai Lam, Susan Plunkett, and Michael Zimmermann for their technical assistance. We also thank MDS Pharma Services for clinical conduct. Financial support was provided by Philip Morris USA, Inc. References 1. K.D. Brunnemann and D. Hoffmann. Analytical studies on tobacco-specific N-nitrosamines in tobacco and tobacco smoke. Toxicology 21: (1991). 2. S.S. Hecht, C.H.B. Chen, R. Young, and D. Hoffmann. Mass spectra of tobacco alkaloid-derived nitrosamines, their metabolites, and related compounds. Beitr. Tabakforsch. Int. 11: (1981). 3. A. Wiernik, A. Christakopoulos, L. Johansson, and I. Wahlberg. Effect of air-curing on the chemical composition of tobacco. Recent Adv. Tob. Sci. 21: (1995). 4. S. Fischer, B. Spiegelhalder, and R. Preußmann. Preformed tobacco-specific nitrosamines in tobacco role of nitrate and influence of tobacco type. Carcinogenesis 10: (1989). 5. International Agency for Research on Cancer Working Group on the Evaluation of Carcinogenic Risks to Humans. Tobacco smoke and involuntary smoking. IARC Monogr. Eval. Carcinog. Risks Hum. 83: (2004). 6. International Agency for Research on Cancer Working Group on the Evaluation of Carcinogenic Risks to Humans Working Group on the Evaluation of Carcinogenic Risks to Humans. Smokeless tobacco and some tobacco-specific N-nitrosamines. IARC Monogr. Eval. Carcinog. Risks Hum. 89: (2007). 7. S.S. Hecht and D. Hoffmann. The relevance of tobacco-specific nitrosamines to human cancer. Cancer Surv. 8: (1989). 8. S.S. Hecht. Biochemistry, biology, and carcinogenicity of tobaccospecific N-nitrosamines. Chem. Res. Toxicol. 11: (1998). 9. P. Upadhyaya, C.L. Zimmerman, and S.S. Hecht. Metabolism and pharmacokinetics of N'-nitrosonornicotine in the patas monkey. Drug Metab. Dispos. 30: (2002). 10. I. Stepanov and S.S. Hecht. Tobacco-specific nitrosamines and their pyridine-n-glucuronides in the urine of smokers and smokeless tobacco users. Cancer Epidemiol. Biomarkers Prev. 14: (2005). 11. S. Feng, S. Kapur, M. Sarkar, R. Muhammad, P. Mendes, K. Newland, and H.J. Roethig. Respiratory retention of nicotine and urinary excretion of nicotine and its five major metabolites in adult male smokers. Toxicol. Lett. 173: (2007). 12. S. Feng, S.E. Plunkett, K. Lam, S. Kapur, R. Muhammad, Y. Jin, M. Zimmermann, P. Mendes, R. Kinser, and H.J. Roethig. A new method for estimating the retention of selected smoke constituents in the respiratory tract of smokers during cigarette smoking. Inhal. Toxicol. 19: (2007). 13. C. Jansson, A. Paccou, and B.G. Osterdahl. Analysis of tobaccospecific N-nitrosamines in snuff by ethyl acetate extraction and liquid chromatography tandem mass spectrometry. J. Chromatogr. A 1008: (2003). 14. S.G. Carmella, S.A. Akerkar, J. Richie, and S.S. Hecht. Intraindividual and interindividual differences in metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3- pyridyl)-1-butanone (NNK) in smokers urine. Cancer Epidemiol. Biomarkers Prev. 4: (1995). 15. M. Meger, I. Meger-Kossien, K. Riedel, and G. Scherer. Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Biomarkers 5: (2000). 16. I. Stepanov and S.S. Hecht. Detection and quantitation of N'- nitrosonornicotine in human toenails by liquid chromatographyelectrospray ionization tandem mass spectrometry. Cancer Epidemiol. Biomarkers Prev. 17: (2008). Manuscript received December 13, 2008; revision received February 6,