Protocol - NNAL in Urine

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NNAL can be measured in urine of people using tobacco products and in urine of non-smokers exposed to secondhand smoke using liquid chromatography - tandem mass spectrometry (LC-MS/MS). 

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: Personal protective equipment (PPE) including eye protection, gloves and suitable protective clothing when used to extract and process samples.




Collect urine sample in a sterile container.  Aliquot 10 mL urine sample to be frozen and stored immediately at -20° C.

Liquid chromatography – tandem mass spectrometry (LC-MS/MS) is the preferred method to accurately measure NNAL in urine. (See source references for details.) The limit of quantitation is 0.25 pg/mL.

Personnel and Training Required

Laboratory training in the use of liquid chromatography and tandem mass spectrometry is required, as well as general laboratory training on the safe use of chemicals and solvents.

Equipment Needs

This method requires high-performance liquid chromatography and tandem mass spectrometry on a triple quadrupole mass spectrometer.

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. 

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). 



Derived Variables


Process and Review

The Tobacco Regulatory Research (TRR) Content Expert Panel (CEP) reviewed the measures in the Tobacco Regulatory Research collection in February 2024.

Guidance from the TRR CEP includes:

  • Replaced protocol
  • New Data Dictionary

Previous version in Toolkit archive (link)

Protocol Name from Source

Determination of NNAL in urine using liquid chromatography - tandem mass spectrometry (LC-MS/MS)


Xia Y, McGuffey JE, Bhattacharyya S, Sellergren B, Yilmaz E, Wang L, Bernert JT. 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. Anal Chem. 2005 Dec 1;77(23):7639-45. doi: 10.1021/ac058027u.PMID: 16316171

Jacob P 3rd, Havel C, Lee DH, Yu L, Eisner MD, Benowitz NL. Subpicogram per milliliter determination of the tobacco-specific carcinogen metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in human urine using liquid chromatography-tandem mass spectrometry. Anal Chem. 2008 Nov 1;80(21):8115-21. doi: 10.1021/ac8009005. Epub 2008 Oct 8. PMID: 18841944, PMC3167662

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
Tobacco Regulatory Research - Host: Biobehavioral
Measure Name

NNAL in Urine

Release Date

May 3, 2024


An assay to measure NNAL, the primary metabolite of NNK.


To measure exposure to tobacco-specific nitrosamine carcinogens.


biomarker, NNAL, urine, tobacco-specific carcinogen, TSNA, tobacco smoke exposure, tobacco product exposure, tobacco chemical exposure, smokeless tobacco exposure, laboratory protocol, second hand smoke, secondhand smoke, second-hand smoke

Measure Protocols
Protocol ID Protocol Name
720402 NNAL in Urine

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