Protocol - Biomarker of exposure to nicotine-containing products - Serum

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Cotinine in serum or plasma is best measured by 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.




Serum or plasma collected by standard procedures is acceptable. At least 3 mL should be provided, to have enough sample volume for a repeat analysis if needed. Samples should be stored at -20° C.

Liquid chromatography – tandem mass spectrometry (LC-MS/MS) is the preferred method to accurately measure cotinine in serum or plasma samples, especially when used to assess secondhand smoke exposure. (See source references for details.) The limit of quantitation is 0.02 ng/mL.

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 on a triple quadrupole mass spectrometer.

Requirement CategoryRequired
Major equipment Yes
Specialized training Yes
Specialized requirements for biospecimen collection No
Average time of greater than 15 minutes in an unaffected individual No
Mode of Administration



Child, Adolescent, Adult


All participants who can provide a blood sample who are 3 years old and older.

Selection Rationale

Cotinine is a major metabolite of nicotine that can be used as a marker for active smoking and as an index for SHS exposure, otherwise known as passive smoking. Cotinine generally is preferred over nicotine for such assessments because of its substantially longer half-life. The estimated half-life of cotinine in plasma is about 15-20 hours; by contrast, the half-life of nicotine is only 0.5-3 hours. Cotinine can be measured in serum, urine, or saliva-the half-life of cotinine in all three fluids is essentially the same (1). It is useful to measure nicotine exposure biomarkers (e.g., cotinine) in multiple matrices such as blood and urine; blood plasma or serum has been used as the fluid of choice for studies requiring a quantitative assessment of nicotine exposure. 

There are other assays (e.g., gas chromatography (GC), gas chromatography mass spectrometry (GC-MS), radioimmunoassay and enzyme-linked immunosorbent assays) used to measure cotinine but liquid chromatography –  tandem mass spectrometry (LC-MS/MS) is preferred, especially when used to assess secondhand smoke exposure.

Liquid chromatography –  tandem mass spectrometry (LC-MS/MS) is the preferred method for determination of cotinine in serum or plasma (see source references 1,2) and other biological fluids, especially for measuring concentrations in non-smokers for exposure to SHS and THS (see source references 1,3).



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 cotinine in serum or plasma using liquid chromatography - tandem mass spectrometry (LC-MS/MS)


Bernert JT, Jacob P 3rd, Holiday DB, Benowitz NL, Sosnoff CS, Doig MV, Feyerabend C, Aldous KM, Sharifi M, Kellogg MD, Langman LJ. Interlaboratory comparability of serum cotinine measurements at smoker and nonsmoker concentration levels: a round-robin study. Nicotine Tob Res. 2009 Dec;11(12):1458-66. doi: 10.1093/ntr/ntp161. Epub 2009 Nov 23. PMID: 19933777, PMC3167685

Sosnoff CS, Caron K, Akins JR, Dortch K, Hunter RE, Pine BN, Feng J, Blount BC, Li Y, van Bemmel DM, Kimmel HL, Edwards KC, Goniewicz ML, Hatsukami DK, de Castro BR, Bernert JT, Arnstein S, Borek N, Deng-Bryant Y, Mishina E, Lawrence C, Hyland A, Hecht SS, Conway KP, Pirkle JL, Wang Serum Concentrations of Cotinine and Trans-3-Hydroxycotinine in US Adults: Results From Wave 1 (2013-2014) of the Population Assessment of Tobacco and Health Study. L. Nicotine Tob Res. 2022 Mar 26;24(5):736-744. doi: 10.1093/ntr/ntab240. PMID: 34897512, PMC8962725.

Jacob P 3rd, Yu L, Duan M, Ramos L, Yturralde O, Benowitz NL. 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. J Chromatogr B Analyt Technol Biomed Life Sci. 2011 Feb 1;879(3-4):267-76. doi: 10.1016/j.jchromb.2010.12.012. Epub 2010 Dec 21. PMID: 21208832, PMC3050598.

General References

Avila-Tang E, Al-Delaimy WK, Ashley DL, Benowitz N, Bernert JT, Kim S, Samet JM, Hecht SS. (2013). Assessing secondhand smoke using biological markers. Tob Control, 22(3): 164-871.

Jarvis, M. J., Russell, M. A. H., Benowitz, N. L., & Feyerabend, C. (1988). Elimination of cotinine from body fluids: Implications for noninvasive measurement of tobacco smoke exposure. American Journal of Public Health, 78, 696-698.

Bernert, J. T., Turner, W. E., Pirkle, J. L., Sosnoff, C. S., Akins, J. R., Waldrep, M. K., et al. (1997). Development and validation of a sensitive method for determination of serum cotinine in smokers and nonsmokers by liquid chromatography / atmospheric pressure ionization tandem mass spectrometry. Clinical Chemistry, 43, 2281-2291.

Protocol ID


Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
Measure Name

Biomarker of exposure to nicotine-containing products

Release Date

May 3, 2024


Cotinine is a major metabolite of nicotine and is an indicator of exposure to nicotine from tobacco or other nicotine containing products.


To assess smoking and environmental tobacco smoke (ETS) exposure by measuring cotinine, a metabolite of nicotine. To screen for tobacco use and quantity and to estimate exposure to secondhand smoke (SHS) and all tobacco exposure. Also used as an outcome measure in smoking cessation trials to determine if an individual has quit smoking.


biomarker of exposure to nicotine-containing products - serum, cotinine, Respiratory, Liquid Chromatography – Tandem mass spectrometry (LC-MS/MS), LC-MS/MS

Measure Protocols
Protocol ID Protocol Name
91704 Biomarker of exposure to nicotine-containing products - Urine
91705 Biomarker of exposure to nicotine-containing products - Saliva
91706 Biomarker of exposure to nicotine-containing products - Serum

There are no publications listed for this protocol.