Protocol - NNAL in Urine
Detailed laboratory protocol for measuring urinary NNAL as used by the Centers for Disease Control and Prevention to analyze urine specimens from the National Health and Nutrition Examination Survey (NHANES).
Because of the sensitive nature of this assay, analysts cannot be actively using tobacco products, and measurements must be performed in a smoke-free environment.
Safety Precautions: Wear eye protection and suitable protective clothing when using this method to extract and process samples.
NNAL is measured by using liquid chromatography linked to tandem mass spectrometry (LC-MS/MS). For "total" NNAL assays, the urine sample is fortified with an NNAL-13C6 internal standard, and then hydrolyzed using β-glucuronidase in incubations for at least 24 hours. The samples are then extracted on a supported liquid extraction cartridge, followed by liquid-liquid extraction and finally cleaned up on a specially-designed solid-phase molecularly-imprinted polymer (MIP) column, after which the analyte is eluted and analyzed by LC/MS/MS, monitoring the m/z 210->180 native, and m/z 216->186 internal standard transition ions. NNAL concentrations are derived from the ratio of the integrated peaks of native to labeled ions by comparison to a standard calibration curve. Free NNAL measurements are conducted in a similar manner, but with the omission of prior enzymatic hydrolysis. Bound NNAL (e.g. NNAL-Gluc) may be estimated from the difference of (Total NNAL - Free NNAL). Prior to assaying each run of unknowns, the results from standard analyses are reviewed for acceptable accuracy, precision and instrument sensitivity. The results from the 20 calibration standards analyzed prior to each run are reviewed daily. Acceptable back-calculated values for standards above the detection limit are typically in the range of nominal concentration ± 10%. The limit of detection is 0.6 pg/mL. Liquid chromatography-tandem mass spectrometry (LC-MS-MS) is the preferred method to accurately measure NNAL in urine samples (see source references for details).
Personnel and Training Required
Laboratory training in the use of liquid chromatography and tandem mass spectrometry is required. All analysts must be CLIA-certified and demonstrate proficiency in the analysis before handling samples.
This method requires high-performance liquid chromatography and tandem mass spectrometry as detailed in the National Health and Nutrition Examination Survey (NHANES) Laboratory Procedures Manual
|Specialized requirements for biospecimen collection||Yes|
|Average time of greater than 15 minutes in an unaffected individual||No|
Mode of Administration
Infant, Toddler, Child, Adolescent, Adult, Senior, Pregnancy
All participants who can provide urine sample. See section 4 of the "CDC Laboratory Procedure Manual for NNAL in Urine."
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) is a major metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), the most potent pulmonary carcinogen of all tobacco-specific n-nitrosamines (TSNA). TSNAs are known to be carcinogenic to many animal species and are believed to be carcinogenic to humans as well. These carcinogens are of special significance because they combine an inherent potent pulmonary carcinogenic potential with a high degree of tobacco-exposure specificity because they can be found only in tobacco and tobacco smoke. Measuring NNAL in urine is a reliable way to determine exposure to NNK for smokers, for nonsmokers exposed to environmental tobacco smoke (ETS), and for people who use smokeless tobacco products (e.g., chewing tobacco, snuff). This protocol was used to measure urinary NNAL in NHANES from 2007 to 2014.
|Human Phenotype Ontology||Triggered by smoking||HP:0025223||HPO|
|caDSR Form||PhenX PX720401 - Nnal In Urine||6238531||caDSR Form|
Process and Review
Protocol Name from Source
National Health and Nutrition Examination Survey (NHANES), Laboratory Procedure Manual, NNAL In Urine, 2011-2012
CDC Laboratory Procedure Manual for NNAL In Urine, Laboratory Procedures Manual
Hecht, S. S. (2002). Human urinary carcinogen metabolites: Biomarkers for investigating tobacco and cancer. Carcinogenesis, 23, 907-922.
Hecht, S. S. (1999). Tobacco smoke carcinogens and lung cancer. Journal of the National Cancer Institute, 91, 1194-1210.
Hecht, S. S., & Hoffmann, D. (1988). Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke. Carcinogenesis, 9, 875-884.
Preston-Martin, S. (1987). N-nitroso compounds as a cause of human cancer. IARC Scientific Publications, 84, 477-484.
Carmella, S. G., Akerkar, S., & Hecht, S. S. (1993). Metabolites of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in smokers’ urine. Cancer Research, 53, 721-724.
Carmella, S. G., Akerkar, S. A., Richie, J. P., Jr., & Hecht, S. S. (1995). Intraindividual and interindividual differences in metabolites of the tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in smokers’ urine. Cancer Epidemiology, Biomarkers & Prevention, 4, 635-642.
Carmella, S. G., Yoder, A., & Hecht, S. S. (2006). Combined analysis of r-1,t-2,3,c-4-tetrahydroxy-1,2,3,4-tetrahydrophenanthrene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in smokers’ plasma. Cancer Epidemiology, Biomarkers & Prevention, 15, 1490-1494.
Xia, Y., McGuffey, J. E., Bhattacharyya, S., Sellergren, B., Yilmaz, E., Wang, L., & Bernert, J. T. (2005). Analysis of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in urine by extraction on a molecularly imprinted polymer column and liquid chromatography/atmospheric pressure ionization tandem mass spectrometry. Analytical Chemistry, 77, 7639-7645.
Xia, Y., & Bernert, J. T. (in press). Stability of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine samples stored at various temperatures. Journal of Analytical Toxicology.
Ashley, D. L., O’Connor, R. J., Bernert, J. T., Watson, C. H., Polzin, G. M., Jain, R. B., et al. (2010). Effect of differing levels of tobacco-specific nitrosamines in cigarette smoke on the levels of biomarkers in smokers. Cancer Epidemiology, Biomarkers & Prevention. doi: 10.1158/1055-9965.EPI-1-0084.
Hecht, S. S., Carmella, S. G., Murphy, S. E., Riley, W. T., Le, C., Luo, X., Mooney, M., & Hatuskami, D. K. (2007). Similar exposure to a tobacco-specific carcinogen in smokeless tobacco users and cigarette smokers. Cancer Epidemiology, Biomarkers & Prevention, 16, 1567-1572.
Hatsukami, D. K., Benowitz, N. L., Rennard, S. I., Oncken, C., & Hecht, S. S. (2006). Biomarkers to assess the utility of potential reduced exposure products. Nicotine Tobacco Research, 4, 600-622.
Bernert, J. T., Pirkle, J. L., Xia, Y., Jain, R. B., Ashley D. L., & Sampson, E. J. (2010). Urine concentrations of a tobacco-specific nitrosamine carcinogen in the U.S. population from secondhand smoke exposure. Cancer Epidemiology, Biomarkers & Prevention, 19(11), 2969-77
Xia, Y., Jain, R., Bernert, J. T., Ashley, D. L., & Pirkle, J. L. (2011). Tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in smokers in the United States: NHANES 2007-2008. Biomarkers, 16(2), 112-119.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX720401010000||Urinary total NNAL concentration in ng/nL||N/A|
NNAL in Urine
February 20, 2015
An assay to measure NNAL, the primary metabolite of NNK.
To measure exposure to tobacco-specific nitrosamine carcinogens.
nnal in urine, biomarker, NNAL, urine, tobacco-specific carcinogen, TSNA, tobacco smoke exposure, tobacco product exposure, tobacco chemical exposure, smokeless tobacco exposure, Centers for Disease Control and Prevention, National Health and Nutrition Examination Survey, NHANES, laboratory protocol, second hand smoke, secondhand smoke, second-hand smoke
|Protocol ID||Protocol Name|
|720401||NNAL in Urine|
Garcia-Cazarin, M.L., Mandal, R.J., Grana, R., Wanke, K.L., Meissner, H. (2020) Host-agent-vector-environment measures for electronic cigarette research used in NIH grants. Tobacco Control. 2020 January; 29(1). doi: 10.1136/tobaccocontrol-2017-054032