Biomonitoring of environmental tobacco smoke (ETS)-related exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

The exposure of non-smokers to the tobacco-specific N-nitrosamine 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a rodent lung carcinogen, was determined in the air of various indoor environments as well as by biomonitoring of non-smokers exposed to environmental tobacco smoke (ETS) under real-life conditions using the urinary NNK metabolites 4-(N-methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and [4-(N-methylnitrosamino)-1-(3-pyridyl)but-1-yl]-beta-O-D-glucosiduronic acid (NNAL-Gluc). NNK was not detectable (<0.5 ng m^-3) in 11 rooms in which smoking did not occur. The mean NNK concentration in 19 rooms in which smoking took place was 17.5 (2.4-50.0) ng m^-3. The NNK levels significantly correlated with the nicotine levels (r=0.856; p< 0.0001). Of the 29 non-smokers investigated, 12 exhibited no detectable NNAL and NNAL-Gluc excretion (<3 pmol day) in their urine. The mean urinary excretion of NNAL and NNAL-Gluc of the 17 remaining non-smokers was 20.3 (<3-63.2) and 22.9 (<3-90.0) pmol day^-1, respectively. Total NNAL excretion (NNAL+NNAL-Gluc) in all non-smokers investigated significantly correlated with the amount of nicotine on personal samplers worn during the week prior to urine collection (r=0.88; <0.0001) and with the urinary cotinine levels (r=0.40; p=0.038). No correlation was found between NNAL excretion and the reported extent of ETS exposure. Average total NNAL excretion in the non-smokers with detectable NNAL levels was 74 times less than in 20 smokers who were also investigated. The cotinine/total NNAL ratios in urine of smokers (9900) and non-smokers (9300) were similar. This appears to be at variance with the ratios of the corresponding precursors (nicotine/NNK) in mainstream smoke (16400) and ETS (1000). Possible reasons for this discrepancy are discussed. The possible role of NNK as a lung carcinogen in non-smokers is unclear, especially since NNK exposure in non-smokers is several orders of magnitude lower than the ordinary exposure to exogenous and endogenous N-nitrosamines and the role of NNK as a human lung carcinogen is not fully understood.     

Nicotine concentrations in urine and saliva of smokers and non-smokers.

Nicotine concentrations were measured in saliva and urine samples collected from 82 smokers and 56 non-smokers after a morning at work. Each subject answered a series of questions related to their recent intentional or passive exposure to tobacco smoke. All non-smokers had measurable amounts of nicotine in both saliva and urine. Those non-smokers who reported recent exposure to tobacco smoke had significantly higher nicotine concentrations (p less than 0.001) than those who had not been exposed; their concentrations overlapped those of smokers who had smoked up to three cigarettes before sampling had the greatest influence on nicotine concentrations (r=0.62 for saliva and r=0.51 for urine). Neither the nicotine for yield of cigarettes nor the self-reported degree of inhalation had any significant effect on nicotine concentrations.     

Cotinine levels and self-reported smoking status in patients attending a bronchoscopy clinic.

The reliability of self-reported smoking behaviour can vary and may result in bias if errors in misclassification vary with outcome. We examined whether self-report was an accurate measure of current smoking status in patients with malignant or non-malignant respiratory disease. Smoking behaviour was assessed by self-report and by analysis of whole blood for cotinine, a biomarker of exposure to cigarette smoke, in 166 patients attending a bronchoscopy clinic. Cotinine levels ranged from 2.5 to >400 ng ml(-1) blood and were higher in self-reported current smokers (173+/-123 ng ml(-1)) than in never smokers (3.7+/-8.7 ng ml(-1)) or ex-smokers (20.5+/-49.0 ng ml(-1)). Cotinine levels in self-reported current smokers increased with the numbers of cigarettes smoked (p=0.06), and levels in smokers and ex-smokers decreased with the reported length of time since the last cigarette (p=0.001). Using a cotinine level of 20 ng ml(-1) and self-report as the gold standard, the sensitivity and specificity for defining current smoking status were 90.2% and 82.4%, respectively. Out of a total of 125 self-reported current non-smokers, 23 (18.4%) had cotinine levels greater than 20 ng ml(-1). Smoking prevalence was significantly underestimated by self-report (24.7%) when compared with that defined using blood cotinine levels (36.1%: p<0.001). Misclassification of current smoking status was particularly high in ex-smokers, in patients without malignant respiratory disease, in men, and in those below the median age. Such differential misclassification may result in bias in studies examining associations between current smoking habits and disease risk.     

Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry

Nicotine is a major addictive compound in cigarette. Its smoke is rapidly and extensively metabolized to several metabolites in human. Cotinine as a major metabolite of nicotine is commonly used as a biomarker to determine active and passive smokers. Cotinine has a longer half-life ( approximately 20 h) compared to nicotine ( approximately 2h). A simple, sensitive, rapid and high throughput GC-MS method was developed for simultaneous quantification of urinary nicotine and cotinine in passive and active smokers. In the sample preparation method, the analytes and internal standard were first basified and followed by liquid-liquid extraction. Upon completion, anhydrous sodium sulphate was added to the solvent mixture to trap moistures. The clear extract obtained was directly injected into GC-MS, operating under selective ion monitoring (SIM) mode. Calibration curves in the range of 0.5-5000 ng/mL of the analytes in urine matrix were established with linear correlation coefficients (r(2)) greater than 0.997. The limit of detection for both nicotine and cotinine were 0.20 ng/mL. The mean recoveries for nicotine and cotinine were 93.0 and 100.4%, respectively. The within- and between-assay accuracies were between 2.1 and 7.9% for nicotine and between 0.7 and 11.1% for cotinine. Within- and between-assay precisions of 3.3-9.5% for nicotine and 3.4-9.8% for cotinine were also achieved. The method can be used in routine assessment and monitoring of active smoking and exposure to environmental tobacco smoke. The applicability of the assay was demonstrated in a small-scale comparison study between smokers and non-smokers.     

Determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in biologic fluids of smokers and non-smokers using liquid chromatography-tandem mass spectrometry: biomarkers for tobacco smoke exposure and for phenotyping cytochrome P450 2A6 activity

The nicotine metabolite cotinine is widely used to assess the extent of tobacco use in smokers, and secondhand smoke exposure in non-smokers. The ratio of another nicotine metabolite, trans-3'-hydroxycotinine, to cotinine in biofluids is highly correlated with the rate of nicotine metabolism, which is catalyzed mainly by cytochrome P450 2A6 (CYP2A6). Consequently, this nicotine metabolite ratio is being used to phenotype individuals for CYP2A6 activity and to individualize pharmacotherapies for tobacco addiction. In this paper we describe a highly sensitive liquid chromatography-tandem mass spectrometry method for determination of the nicotine metabolites cotinine and trans-3'-hydroxycotinine in human plasma, urine, and saliva. Lower limits of quantitation range from 0.02 to 0.1ng/mL. The extraction procedure is straightforward and suitable for large-scale studies. The method has been applied to several thousand biofluid samples for pharmacogenetic studies and for studies of exposure to low levels of secondhand smoke. Concentrations of both metabolites in urine of non-smokers with different levels of secondhand smoke exposure are presented.     

Cotinine levels and self-reported smoking status in patients attending a bronchoscopy clinic

The reliability of self-reported smoking behaviour can vary and may result in bias if errors in misclassification vary with outcome. We examined whether self-report was an accurate measure of current smoking status in patients with malignant or non-malignant respiratory disease. Smoking behaviour was assessed by self-report and by analysis of whole blood for cotinine, a biomarker of exposure to cigarette smoke, in 166 patients attending a bronchoscopy clinic. Cotinine levels ranged from 2.5 to &gt;400 ng ml^?1 blood and were higher in self-reported current smokers (173±123 ng ml^?1) than in never smokers (3.7±8.7 ng ml^?1) or ex-smokers (20.5±49.0 ng ml^?1). Cotinine levels in self-reported current smokers increased with the numbers of cigarettes smoked (p=0.06), and levels in smokers and ex-smokers decreased with the reported length of time since the last cigarette (p=0.001). Using a cotinine level of 20 ng ml^?1 and self-report as the gold standard, the sensitivity and specificity for defining current smoking status were 90.2% and 82.4%, respectively. Out of a total of 125 self-reported current non-smokers, 23 (18.4%) had cotinine levels greater than 20 ng ml^?1. Smoking prevalence was significantly underestimated by self-report (24.7%) when compared with that defined using blood cotinine levels (36.1%: p&lt;0.001). Misclassification of current smoking status was particularly high in ex-smokers, in patients without malignant respiratory disease, in men, and in those below the median age. Such differential misclassification may result in bias in studies examining associations between current smoking habits and disease risk.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

Factors of humoral resistance in workers exposed to vinyl chloride with a view to smoking habits

Serum samples were assayed in 110 workers (59 smokers and 51 non-smokers) at PVC manufacturing factory, the results being compared with those obtained in a group of age-matched healthy controls. Non-smokers had significantly higher levels of immunoglobulins (IgG, IgA, IgM), while in smokers there was an increase in IgM only. Lysozyme levels (LYS) were elevated in all exposed subjects, but there was a highly significant decrease in the total protein (TP) content. Alfa-2-macroglobulin (A2M) and orosomucoid (ORO) were also elevated in exposed workers. A significant increase was found in ceruloplasmin (CPL), with smokers having higher levels than non-smokers. No difference was detected between the examined groups in transferrin (TRF) and alfa-1-antitrypsin (A1AT). Exposure duration did not correlate with any of the assayed parameters. The positive correlation of IgA levels and inverse correlation of CPL with age in the control group could not be confirmed in the exposed subjects where a significant inverse correlation between age and AlAT levels, which was found to be positive in exposed non-smokers, proved to be inverse in the subgroup of smokers. The authors discuss possible explanations of the reported findings.     

Simultaneous estimation of nicotine and cotinine levels in biological fluids using high-resolution capillary-column gas chromatography combined with solid phase extraction work-up

A rapid and sensitive capillary gas-chromatographic method with nitrogen-sensitive detection is reported for the simultaneous analysis of nicotine and cotinine levels occurring in the plasma, saliva, and urine of regular tobacco smokers. The proposed assay has a linear output, has satisfactory accuracy over the range of concentrations of both amines encountered in active smokers, and has also been successful in the analysis of the urine samples of passive smokers. Its lower limit of sensitivity is 0.2 ng of nicotine and 0.5 ng of cotinine per ml of plasma or saliva or per 100 l of urine.The beneficial characteristics of the presented method were achieved by the combination of solid phase extraction of 0.1–1.0 ml of fluid specimens, capillary column gas chromatography with splitless injection and nitrogen sensitive detection, and the use of separate, structurally analogous compounds as internal standards for nicotine. The suitability of the assay is shown by plasma concentration-time curves of nicotine and cotinine in a steady smoker during a 24 hours period.     

Cigarette smoking reduces human salivary eicosanoids.

The effect of cigarette smoking on salivary eicosanoid levels was investigated in 10 smoker and 10 non-smoker volunteers. The smokers consumed an average of 20 cigarettes/day for the past 5 years or longer. The smoking status was validated by salivary cotinine level. Eicosanoids were extracted from saliva with ethanol, and the radioimmunoassay was performed to determine the concentrations of four major eicosanoids, i.e. prostaglandin E2 (PGE2), PGF2 alpha, 6-sulphidopeptide-containing leukotrienes (LTs) and 12-hydroxyeicosatetraenoic acid (12-HETE). The levels of PGE2, PGF2 alpha, and LTs were significantly lower in the saliva of smokers as compared to that of the non-smokers (1.74 +/- 0.32 vs 2.41 +/- 0.64, p = 0.006; 0.36 +/- 0.12 vs 0.54 +/- 0.18, p = 0.04; 2.24 +/- 0.96 vs 4.92 +/- 1.29, p = 0.006; mean +/- SD, ng/ml saliva). No significant differences were found in the levels of 12-HETE between the two groups. The results suggest that cigarette smoking reduces the concentrations of both the cyclooxygenase and 5-lipoxygenase products in saliva.     

S100B and brain natriuretic peptide predict functional neurological outcome after intracerebral haemorrhage

Colorimetric test strip assays are a convenient and inexpensive means for the determination of cotinine in human urine because they can be performed in a nonlaboratory environment using a trained technician. Four hundred human urine samples were separated into four categories: (1) heavy smokers (>20 cigarettes smoked per day), (2) light smokers (<20 cigarettes smoked per day), (3) non-smokers, and (4) vegetarian non-smokers. Samples were evaluated by a gas chromatography/mass selective detector (GC/MSD) method as a reference and using NicCheck I? (DynaGen, Inc.). Colour intensity can range from 0 (no colour) to 14 (deep pink). Qualitative values were assigned as negative (0), low (1-6) and high (7-14). Comparison of the test strip and GC/MSD results showed: (1) 43 (10.75%) false negatives using the criterion of a GC/MSD cotinine level above 200 ng ml^-1 and test strip reading of 0, (2) 31 (7.75%) false positives using the criterion of a GC/MSD cotinine level below 1 ng ml^-1 and a test strip reading of 1 or greater, and (3) no correlation between the test strip and GC/MSD results (r = 0.597, p < 0.05). The fact that the colorimetric reaction is sensitive to many nicotine metabolites and/or heterocyclic amine structures whereas the GC/MSD method measures nicotine and cotinine selectively might explain the false positive results. False negative results were likely to be due to a lack of sensitivity of the test strip.     

Gas chromatographic analysis of nicotine and cotinine in hair

Non-invasive validation of cigarette- or cigar-smoking behaviour is necessary for large population studies. Urine or saliva samples can be used for confirmation of recent nicotine intake by analysis of cotinine, the major metabolite of nicotine. However, this test is not suitable for validation of survey data, since the quantification of cotinine in saliva only reflects nicotine exposure during the preceding week. To validate information on tobacco use, we investigated hair samples for quantifying nicotine and cotinine by gas chromatography—mass spectrometry. Hair (about 50–100 mg) was incubated in 1 M sodium hydroxide at 100°C for 10 min. After cooling, samples were extracted by diethyl ether, using ketamine as an internal standard. Drugs were separated on a 12-m BP-5 capillary column, and detected using selected-ion monitoring (m/z 84, 98 and 180 for nicotine, cotinine and ketamine, respectively). Hair from non-smokers and smokers contained nicotine and cotinine. Although it is difficult to determine an absolute cut-off concentration, more than 2 ng of nicotine per milligram of hair can be used to differentiate smokers from non-smokers. Some applications of this technique are developed to determine the status of passive smokers, the gestational exposure in babies and the pattern of an individual's nicotine use by cutting strands of hair into sections of one-month intervals.     

Serum Clara cell protein (CC16) in healthy young smokers

The CC16 microprotein is the main secretory product of Clara cells, which are epithelial cells lining lung airways. In crossing through the bronchoalveolar/blood barrier, CC16 diffuses passively into plasma. Serum CC16 (sCC16) has recently been proposed as a biomarker for detecting Clara cell impairments. The aim of this study was to assess if sCC16 concentrations are reduced in a group of healthy young smokers. A group of 118 healthy young males volunteered to take part in the study. Each subject answered a questionnaire, and provided blood and urine samples. Serum CC16, urinary cotinine and creatinine were measured. Median serum CC16 concentrations were lower in smokers than in non-smokers (11.3 mug l^-1 vs 14.6 mug l^-1; p = 0.005; N = 89 and 29, respectively) but did not correlate with either the daily or the life-time cigarette consumption, or with urinary cotinine concentrations. sCC16 did not correlate with age or body mass index in the whole study population or in the groups of smokers and non-smokers. These results suggest the reduction in sCC16 concentrations in a group of healthy young smokers may be an early effect of cigarette smoking.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers (p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

Measurement of 7 methyl and 7 2 hydroxyethyl guanine DNA adducts in white blood cells of smokers and non smokers

The goal of the present study was to measure the levels of 7-methylguanine and 7-(2- hydroxyethyl)guanine DNA adducts in human white blood cells in relation to smoking. DNA was isolated from samples of 11 smokers and eight non-smokers. The 32P-postlabelled 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts were analysed by thin-layer chromatography (TLC) combined with a high pressure liquid chromatography (HPLC) assay. In smokers the mean 7-methylguanine and 7-(2-hydroxyethyl)guanine levels were 32.3 +/- 7.1 and 6.6 +/- 2.3 adducts per 108 nucleotides respectively. The corresponding values in non-smokers were 25.0 +/- 7.0 and 3.7 +/- 2.4 adducts per 108 nucleotides. There were significantly higher levels of 7-methylguanine and 7-(2-hydroxyethyl)guanine adducts in WBC in smokers than in non-smokers ( p = 0.041; p = 0.018), respectively. A positive correlation between 7-methylguanine and 7-(2-hydroxyethyl)guanine levels was observed.     

Adolescent smokers seen in general practice: health,lifestyle, physical measurements,and response to antismoking advice.

OBJECTIVE--To compare physical, lifestyle, and health characteristics of adolescent smokers and non-smokers and their initial response to anti-smoking counselling. DESIGN--Adolescents aged 13, 15, and 17 years were identified from age-sex registers and invited by letter for a general practice health check. SETTING--Three general practices in the MRC general practice research framework. MAIN OUTCOME MEASURES--Blood pressure, body mass index, saliva cotinine concentration, peak flow rate, alcohol consumption, exercise, duration of sleep, and stated persistent health problems. RESULTS--73% of the adolescents (491) attended for the health check. A total of 68 (14%) were regular smokers. By age 17 those who smoked regularly had a significantly lower systolic blood pressure than those who had never smoked regularly (by 6 mm Hg; p = 0.025) despite a significantly higher body mass index (by 1.5; p <0.001) [corrected]. Cotinine concentrations increased with smoking exposure, from 0.7 ng/ml when no family member smoked to 155 ng/ml in active smokers of six or more cigarettes a week. Significantly more regular smokers than never regular smokers drank greater than or equal to 8 g alcohol a day (chi 2 = 15.2 adjusted for age and sex p less than 0.001); regular smokers exercised less (1.0 hrs/week in boys and 0.8 hrs/week in girls v 3.4 hrs/week in boys and 2.2 hrs/week in girls; p less than 0.001) and slept less (8.0 hrs/night v 8.5 hrs/night at age 17; p less than 0.005). Persistent health problems, mostly asthma or allergic symptoms, were reported by 25% (17/68) of the smokers and 16% (60/381) of the non-smokers. Of the smokers given counselling, 60% (26/43) made an agreement with the practice doctor or nurse to give up smoking. CONCLUSION--General practice is an appropriate setting for adolescents to receive advice on healthy lifestyle, which should not focus solely on smoking.     

Verification of smoking history in patients after infarction using urinary nicotine and cotinine measurements.

Urinary concentrations of nicotine and its major metabolite cotinine were measured in volunteers whose smoking habits were known to test the reliability of the measurements as indicators of current smoking. In the non-smokers detectable concentrations were always below the confidence limits set for the method, while in smokers the concentrations were always above these limits. After subjects stopped smoking cotinine appeared in the urine for longer than nicotine and was still detectable at least 36 hours after the last cigarette had been smoked. When this method was used to verify the smoking histories given by patients attending an infarction clinic it was estimated that 46-53% of previous smokers had actually stopped smoking compared with the 63% who said that they had done so. It is suggested that simultaneous assays of urinary nicotine and cotinine may be a useful means of verifying patients'' current smoking habits.     

Validation of an assay for the determination of cotinine and 3-hydroxycotinine in human saliva using automated solid-phase extraction and liquid chromatography with tandem mass spectrometric detection

The validation of a high-performance liquid chromatographic method for the simultaneous determination of low level cotinine and 3-hydroxycotinine in human saliva is reported. Analytes and deuterated internal standards were extracted from saliva samples using automated solid-phase extraction, the columns containing a hyper cross-linked styrene–divinylbenzene copolymer sorbent, and analysed by reversed-phase liquid chromatography with tandem mass spectrometric detection (LC–MS–MS). Lower limits of quantitation of 0.05 and 0.10 ng/ml for cotinine and 3-hydroxycotinine, respectively, were achieved. Intra- and inter-batch precision and accuracy values fell within ±17% for all quality control samples, with the exception of quality control samples prepared at 0.30 ng/ml for 3-hydroxycotinine (inter-day precision 21.1%). Results from the analysis of saliva samples using this assay were consistent with subjects’ self-reported environmental tobacco smoke (ETS) exposures, enhancing the applicability of cotinine as a biomarker for the assessment of low level ETS exposure.     

Clinical considerations in study designs that use cotinine as a biomarker.

Subjects enrolled in studies are not always screened for routine habits such as smoking. Personal history is not always reliable and therefore an objective biomarker is necessary to screen for smokers. The objectives of this article were to review the metabolism of nicotine and other metabolic considerations associated with smoking; to review some of the routine methods used to assess exposure to nicotine-containing products; to revisit cotinine breakpoints utilized to distinguish smokers from non-smokers during screening for clinical trials; to assess the utility of screening questions regarding smoking practices; and to recommend standards for clinical pharmacology studies. The results indicated that cotinine levels serve as a useful biomarker of tobacco exposure; racial issues may be clinically relevant in determining smoking status; cessation of smoking should occur at least 14 days prior to the start of the study; adverse effects from nicotine withdrawal such as craving, hunger and weight gain may persist for more than 6 months; potential metabolic interactions via cytochrome P2A6 and P1A2 need to be considered when designing a study; and the use of a single calibrator as a breakpoint is acceptable if a categorical outcome such as 'smoker' versus 'non-smoker' is desired. Nicotine from food products is not expected to impact assay sensitivity or to be clinically relevant; a serum cotinine concentration of 10 ng ml(-1) be employed as a breakpoint for non-smokers versus smokers; other non-invasive alternatives are collection of urine, saliva, or hair (with suggested breakpoints of 200 ng ml(-1), 5 ng ml(-1) and 0.3 ng mg(-1), respectively; screening questions be accompanied by testing for cotinine; and the inclusion of smokers in studies should be considered once the impact of smoking on the targeted population is understood.