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

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A biospecimen is collected from the respondent to measure cotinine, a marker of either smoking or of environmental tobacco smoke exposure. One of the most common methods is via a urine sample obtained from the respondent. Cotinine in urine is measured by gas chromatography mass spectrometry (GC-MS).

Specific Instructions

Caffeine may interfere with the accuracy of the results. During the gas chromatography analysis, this interference can be minimized by ramping up the temperature and eluting the caffeine later than the cotinine.


The respondent is asked to urinate in a sterile 90-mL urine specimen container. Children above the age of 10 and adults can self-collect the urine. The specimen should be refrigerated immediately after collection. Aliquot 3mLs into a 4 mL cryovial. Freeze the cryovial at -20°C until analysis. If the samples need to be stored greater than 1 year freeze at -80°C.

Gas chromatography mass spectrometry (GC-MS) is the preferred method to accurately measure cotinine in urine samples. (See source references for details.) The limit of detection is 0.20 ng/mL. A result of 100 ng/mL urinary cotinine or more is indicative of an active smoker.



Personnel and Training Required

Urine samples can be self-collected by children aged 10 or older with adequate instructions. A laboratory technician trained to process and analyze biological specimens is necessary to analyze urine samples.

Equipment Needs

Standard urine collection supplies that have been sterilized. Laboratory supplies and instruments are needed to measure cotinine in biological fluids. Biological samples may be shipped using appropriate shipping procedures to laboratories that specialize in these types of analysis.

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, Senior, Pregnancy


Ages 3 and up

Selection Rationale

Urine is easily collected and with the proper laboratory equipment, cotinine can be measured accurately from a sterile urine sample. Urinary cotinine is a frequently measured biomarker and produces more quantifiable results than other metabolites.

There are other assays (e.g., radioimmunoassay and enzyme-linked immunosorbent assays) used to measure cotinine but gas chromatography mass spectrometry (GC-MS) is preferred.


Chinese, English

Logical Observation Identifiers Names and Codes (LOINC) Resp urine assay smoke exp proto 62641-6 LOINC
caDSR Form PhenX PX091701 - Urine Assay For Tobacco Smoke Exposure 5970459 caDSR Form
Derived Variables


Process and Review

The Expert Review Panel #6 (ERP 6) reviewed the measures in the Respiratory domain.

Guidance from ERP 6 includes:

  • Revised descriptions of the measure
  • Changed the name of the measure from "Urine Assay for Tobacco Smoke Exposure" to "Biomarker of Exposure to Nicotine-containing Products"

Previous version can be found in the Toolkit archive (link)

Protocol Name from Source

Man, C. N., et al, Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry, J CHROMATOGR, 2006


Man, C. N., L.-H. Gam, Ismail, S., Lajis, R., Awang, R. 2006. Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry. J of Chromatography B 844(2): 322-327.

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.

Benowitz, N.L. (1999). Biomarkers of Environmental Tobacco Smoke Exposure. Environ Health Perspect. 107(Suppl 2):3 49-355.

Benowitz, N. L. (1996). Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev, 18(2), 188-204.

Dhar, P. (2004). Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods. J Pharm Biomed Anal, 35(2004), 155-168.

Kuo, H.W., Yang, J.S., Chiu, M.C. (2002). Determination of urinary and salivary cotinine using gas and liquid chromatography and enzyme-linked immunosorbent assay. J Chromatogr B Analyt Technol Biomed Life Sci. 768(2):297-303.

Protocol ID


Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX091701010000 Concentration of urinary cotinine in ng/mL. Variable Mapping
Measure Name

Biomarker of exposure to nicotine-containing products

Release Date

May 12, 2010


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 - urine, 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

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