A liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the determination of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in urine of non-smokers and smokers.

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials and, therefore, occur ubiquitously in the environment and also in tobacco smoke. Since some PAH have been classified as carcinogens, it is important to have access to suitable analytical methods for biomarkers of exposure to this class of compounds. Past experience has shown that measuring a profile of PAH metabolites is more informative than metabolites of a single PAH. Assessment of environmental and smoking-related exposure levels requires analytical methods with high sensitivity and specificity. In addition, these methods should be fast enough to allow high throughput. With these pre-conditions in mind, we developed and validated a high-performance liquid chromatographic method with tandem mass spectrometric detection (LC-MS/MS) for the determination of phenolic metabolites of naphthalene, fluorene, phenanthrene and pyrene in urine of smokers and non-smokers. Sample work-up comprised enzymatic hydrolysis of urinary conjugates and solid-phase extraction on C18 cartridges. The method showed good specificity, sensitivity, and accuracy for the intended purpose and was also sufficiently rapid with a sample throughput of about 350 per week. Application to urine samples of 100 smokers and 50 non-smokers showed significant differences between both groups for all measured PAH metabolites, and strong correlations with markers of daily smoke exposure in smoker urine. Urinary levels were in good agreement with previously reported data using different methodologies. In conclusion, the developed LC-MS/MS method is suitable for the quantification of phenolic PAH metabolites of naphthalene, fluorene, phenanthrene, and pyrene in smoker and non-smoker urine.

Urinary excretion of phenolic polycyclic aromatic hydrocarbons (OH-PAH) in nonsmokers and in smokers of cigarettes with different ISO tar yields.

Polycyclic aromatic hydrocarbons (PAH) are products of the incomplete combustion of organic materials, and they occur ubiquitously in the environment. They are also present in tobacco smoke. Some PAH have been classified as carcinogens; therefore, it is important to develop and assess suitable biomarkers for PAH exposure. A high-performance liquid chromatographic method with fluorescence detection was developed to determine 1- and 2-hydroxynaphthalene (1- and 2-OH-Nap), 2-hydroxyfluorene (2-OH-Flu), 2-/3-hydroxyphenanthrene (2-/3-OH-Phe), 1-/9-hydroxyphenanthrene (1-/9-OH-Phe), and 1-hydroxypyrene (1-OH-Pyr) in human urine. The method is sensitive (LOQ ranging from 0.01 ng/mL for 1-OH-Pyr to 1 ng/mL for the naphthols), precise (interday precision ranging from 1.4 to 6.9%), and accurate (97-106%). The method was applied to 108 urine samples from 25 nonsmokers and 83 smokers. Smokers excreted significantly higher amounts of 1-OH-Nap (16.1 vs. 2.9 microg/24 h), 2-OH-Nap (20.9 vs. 9.7 microg/24 h), 2-OH-Flu (1.87 vs. 0.75 microg/24 h), 2-/3-OH-Phe (0.73 vs. 0.50 microg/24 h), 1-/9-OH-Phe (0.66 vs. 0.35 microg/24 h), and 1-OH-Pyr (0.36 vs. 0.20 microg/24 h) compared to nonsmokers. In conclusion, the method is suitable for discriminating PAH exposure between different ISO tar yield cigarette smokers, and it may be applicable in evaluating future potential reduced exposure tobacco products.

Biotransformation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in lung tissue from mouse, rat, hamster, and man.

Exposure to the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is considered to be an important etiological risk factor for lung cancer in tobacco users. The metabolism of NNK via carbonyl reduction to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), alpha-hydroxylation to form both DNA methylating and pyridyloxobutylating intermediates, and detoxification by pyridyl N-oxidation and glucuronide formation are well-characterized in laboratory animals but less so in man. The in vitro kinetics of 0.03-250 microM [5-(3)H]NNK metabolism were determined under identical experimental conditions using female A/J mouse, male Fischer 344 rat, female Syrian golden hamster, and human lung tissue explants in tissue culture. The concentration-dependent percentage contribution of the three major pathways of NNK metabolism (carbonyl reduction, alpha-hydroxylation, and N-oxidation) showed large interspecies variation. Quantitatively, in mouse, carbonyl reduction to NNAL increased steadily with an increasing substrate concentration (10-74% total NNK metabolism), while concurrent decreases occurred in end products of alpha-hydroxylation (60 to 18%) and N-oxidation (42 to 5%). In rat lung, there were no apparent concentration-dependent trends (NNAL, 42 +/- 4%; alpha-hydroxylation, 35 +/- 2%; and N-oxidation, 24 +/- 3%). In hamster lung, a clear concentration-dependent increase in the contribution of NNAL to total NNK metabolism (from 47 to 87%) was paralleled by a steady decline in end products of alpha-hydroxylation (31 to 11%) and N-oxidation (22 to 2%). Human lung metabolism showed no concentration-dependent tendencies (NNAL, 89 +/- 1%; alpha-hydroxylation, 8.8 +/- 1.1%; and N-oxidation, 2.1 +/- 0.3%). The major alpha-hydroxylation product in human lung was 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), thus supporting the potential pyridyloxobutylation of lung DNA. Metabolism to 4-(3-pyridyl)-4-oxobutanoic acid (keto acid), which could result in lung DNA methylation, was only sporadically seen in human lung but present to a far greater extent in rodent lung. No evidence for glucuronidation was found in any species. Generally, the rate of formation of all NNK metabolites showed two different enzyme kinetics, resulting in large differences between apparent K(m) and V(max) values in the low (up to 2.8 microM) and high substrate concentration ranges. The metabolism of NNK by alpha-hydroxylation is considerably lower in human lung as compared to that observed in rodent species, suggesting that extrapolation of in vitro rodent data to man may result in invalid conclusions about the capacity of the human lung to activate NNK under realistic conditions of NNK exposure expected to occur in man.

Comparison of environmental tobacco smoke (ETS) concentrations generated by an electrically heated cigarette smoking system and a conventional cigarette.

Smoking conventional lit-end cigarettes results in exposure of nonsmokers to potentially harmful cigarette smoke constituents present in environmental tobacco smoke (ETS) generated by sidestream smoke emissions and exhaled mainstream smoke. ETS constituent concentrations generated by a conventional lit-end cigarette and a newly developed electrically heated cigarette smoking system (EHCSS) that produces only mainstream smoke and no sidestream smoke emissions were investigated in simulated "office" and "hospitality" environments with different levels of baseline indoor air quality. Smoking the EHCSS (International Organisation for Standardization yields: 5 mg tar, 0.3 mg nicotine, and 0.6 mg carbon monoxide) in simulated indoor environments resulted in significant reductions in ETS constituent concentrations compared to when smoking a representative lit-end cigarette (Marlboro: 6 mg tar, 0.5 mg nicotine, and 7 mg carbon monoxide). In direct comparisons, 24 of 29 measured smoke constituents (83%) showed mean reductions of greater than 90%, and 5 smoke constituents (17%) showed mean reductions between 80% and 90%. Gas-vapor phase ETS markers (nicotine and 3-ethenylpyridine) were reduced by an average of 97% (range 94-99%). Total respirable suspended particles, determined by online particle measurements and as gravimetric respirable suspended particles, were reduced by 90% (range 82-100%). The mean and standard deviation of the reduction of all constituents was 94 +/- 4%, indicating that smoking the new EHCSS in simulated "office" and "hospitality" indoor environments resulted in substantial reductions of ETS constituents in indoor air.

Relationship between machine-derived smoke yields and biomarkers in cigarette smokers in Germany.

In order to determine whether smokers of cigarettes in the contemporary yield ranges of the German market (0.1-1.0mg nicotine, 1-10mg tar) differ in their actual exposure to various smoke constituents, we performed a field study with 274 smokers and 100 non-smokers. The following biomarkers were determined: In 24-h urine: Nicotine equivalents (molar sum of nicotine, cotinine, trans-3'-hydroxycotinine and their respective glucuronides), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL, metabolite of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, NNK), 3-hydroxypropylmercapturic acid (metabolite of acrolein), trans,trans-muconic acid, S-phenylmercapturic acid (metabolites of benzene), 1-hydroxypyrene (metabolite of pyrene); in saliva: Cotinine and trans-3'-hydroxycotinine; in exhaled air: Carbon monoxide; in blood: Methyl-, hydroxyethyl-, cyanoethyl- (biomarker of acrylonitrile) and carbamoylethylvaline (biomarker of acrylamide) hemoglobin adducts. All biomarkers were found to be significantly higher in smokers compared to non-smokers and showed strong correlations with the daily cigarette consumption. Biomarker levels and per cigarette increases in smokers were at most weakly related to the machine-derived smoke yields. It is concluded that machine-derived yields of cigarettes from the contemporary German cigarette market have little or no impact on the actual smoking-related exposure determined by suitable biomarkers.

Determination of three carcinogenic aromatic amines in urine of smokers and nonsmokers.

Aromatic amines (arylamines) such as o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl occur in the environment and are constituents of tobacco smoke. Human exposure to these aromatic amines has long been associated with an elevated risk of bladder cancer. A validated, specific, and sensitive method for measuring o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in cigarette smokers and nonsmokers was developed. The method uses acid hydrolysis of the arylamine conjugates in urine, extraction with n-hexane, derivatization with pentafluoropropionic anhydride, and subsequent analysis with gas chromatography combined with mass spectrometry using negative ion chemical ionization. The limits of detection were 4 ng/L for o-toluidine and 1 ng/L for 2-aminonaphthalene and 4-aminobiphenyl. Smokers (N = 10) excreted significantly higher amounts of o-toluidine (204 versus 104 ng/24 h), 2-aminonaphthalene (20.8 versus 10.7 ng/24 h), and 4-aminobiphenyl (15.3 versus 9.6 ng/24 h) than nonsmokers (N = 10). Urinary arylamine excretion in smokers was associated with the extent of smoking as assessed by daily cigarette consumption, urinary excretion of nicotine equivalents (nicotine plus its five major metabolites), cotinine in saliva, and carbon monoxide in exhaled breath. All nonsmokers investigated had quantifiable amounts of o-toluidine, 2-aminonaphthalene, and 4-aminobiphenyl in their urine, confirming that other environmental sources of exposure to these compounds also occur. In conclusion, the analytical method is suitable for measuring short-term exposure to arylamines in urine of non-occupationally exposed smokers and nonsmokers.

Influence of smoking charcoal filter tipped cigarettes on various biomarkers of exposure.

Charcoal (CC) filters of cigarettes are known to significantly reduce a series of volatile constituents in mainstream smoke, including reactive alpha,beta-unsaturated aldehydes such as acrolein and crotonaldehyde. We performed a randomized, crossover, 2-wk brand-switching study with 39 smokers. Twenty of the subjects smoked cellulose acetate (CA) filter tipped cigarettes during wk 1 of the study; the remaining 19 subjects smoked CC filter tipped cigarettes during wk 1. In wk 2, the subjects switched to the corresponding brand with the other filter type, with similar smoking machine-derived tar and nicotine yields. Daily cigarette consumption, carbon monoxide in exhaled breath, salivary cotinine, and urinary nicotine equivalents (molar sum of nicotine plus five major metabolites) did not change significantly when switching to the cigarettes with the other filter type. Urinary excretion rates of 3-hydroxy-1-methylpropylmercapturic acid (metabolite of crotonaldehyde), monohydroxybutenylmercapturic acid (metabolite of 1,3-butadiene), and S-phenylmercapturic acid (metabolite of benzene) were significantly lower when smoking CC compared to CA filter tipped cigarettes. The reduction in amount of 3-hydroxypropylmercapturic acid (metabolite of acrolein) was of borderline significance. Other mercapturic acids and thioethers (the latter is a summary parameter that indicates the exposure to electrophilic compounds) were not or were only slightly reduced upon smoking CC filter tipped cigarettes. We conclude that smoking CC filter tipped cigarettes does not change the uptake of carbon monoxide and nicotine when compared to CA filter tipped cigarettes with similar tar and nicotine yields, but significantly reduces the exposure to toxicologically relevant smoke constituents such as acrolein, crotonaldehyde, 1,3-butadiene, and benzene.