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

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This is the laboratory protocol for measuring serum cotinine, which is used by the Centers for Disease Control and Prevention and for National Health and Nutrition Examination Survey (NHANES) cotinine analyses.

Specific Instructions



Serum cotinine is measured by high-performance liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry (LC APCI MS/MS). Briefly, a 0.5-ml aliquot of the serum sample is equilibrated with a cotinine-D3 internal standard for 20 min, and then applied to a basified solid-phase extraction column. Cotinine is extracted using methylene chloride, the organic extract is concentrated, and the residue is injected onto a C-18 HPLC column. The eluant from these injections is monitored by APCI-MS/MS, and the m/z 80 daughter ion from the m/z 177 quasi-molecular ion is quantitated. Additional ions for the internal standard and for confirmation are also monitored. Cotinine concentrations are derived from the ratio of native-to-labeled cotinine in the sample by comparisons to a standard curve. The limit of detection (LOD) is 15 pg/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 as detailed in the attached document "CDC Laboratory Procedure Manual for COTININE IN SERUM."

Requirement CategoryRequired
Major equipment No
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. For that reason, serum was chosen as the matrix for the NHANES cotinine analyses. This protocol was used to measure serum cotinine in NHANES from 2002 to 2012 (2,3).



caDSR Common Data Elements (CDE) Serum Cotinine Assay Concentration Level Code 6454566 CDE Browser
Derived Variables


Process and Review

Not applicable.

Protocol Name from Source

National Health and Nutrition Examination Survey (NHANES), Cotinine Analysis, 2011-2012


CDC Laboratory Procedure Manual for Cotinine in Serum

Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS). (2011-2012). National Health and Nutrition Examination Survey Anthropometry Procedures Manual. Hyattsville, MD: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention

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
PX091703010000 Concentration of serume cotinine in ng/mL. N/A
Measure Name

Biomarker of exposure to nicotine-containing products

Release Date

November 28, 2017


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.


cotinine, Centers for Disease Control and Prevention, CDC, National Health and Nutrition Examination Survey, NHANES, Respiratory

Measure Protocols
Protocol ID Protocol Name
91701 Biomarker of exposure to nicotine-containing products - Urine
91702 Biomarker of exposure to nicotine-containing products - Saliva
91703 Biomarker of exposure to nicotine-containing products - Serum

There are no publications listed for this protocol.