Protocol - NNAL in Urine

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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).

Specific Instructions

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.

Equipment Needs

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

Requirement CategoryRequired
Major equipment Yes
Specialized training Yes
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."

Selection Rationale

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
Derived Variables


Process and Review

Not applicable.

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

General References

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.

Protocol ID


Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX720401010000 Urinary total NNAL concentration in ng/nL N/A
Tobacco Regulatory Research - Host: Biobehavioral
Measure Name

NNAL in Urine

Release Date

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

Measure Protocols
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