Protocol - Biomarker of exposure to nicotine-containing products - Urine
Description
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 best measured by liquid chromatography - tandem mass spectrometry (LC-MS/MS).
Specific Instructions
Cotinine is excreted as both “free” cotinine and as its glucuronide conjugate. Most methods measure free cotinine. If total (free + glucuronide) concentrations are needed, the urine sample can be treated with a glucuronidase enzyme to deconjugate the glucuronides prior to analysis.
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
Availability
Protocol
Collect urine sample in a sterile container. Aliquot a minimum of 5 mL urine sample to be frozen and stored immediately at -20° C. However, if it can?t be promptly frozen, may be left at room temperature for extended periods; acidification with sodium bisulfate to pH= 2-3 is advantageous to stabilize the sample.
Liquid chromatography – tandem mass spectrometry (LC-MS/MS) is the preferred method to accurately measure cotinine in urine samples, especially when used to assess secondhand smoke exposure. (See source references for details.) The limit of quantitation is 0.05 ng/mL.
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. 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
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. This method requires high-performance liquid chromatography and tandem mass spectrometry on a triple quadrupole mass spectrometer.
Requirements
| Requirement Category | Required |
|---|---|
| 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
Bioassay
Lifestage
Child, Adolescent, Adult, Senior, Pregnancy
Participants
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., 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.
Determination of cotinine in urine using liquid chromatography – tandem mass spectrometry (LC-MS/MS) is the preferred method for determination of cotinine in urine (see source reference 1) and other biological fluids, especially for measuring concentrations in non-smokers for exposure to SHS and THS (see source reference 2).
Language
English
Standards
| Standard | Name | ID | Source |
|---|
Derived Variables
None
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 urine using liquid chromatography - tandem mass spectrometry (LC-MS/MS)
Source
1. Wang L, Bernert JT, Benowitz NL, Feng J, Jacob P 3rd, McGahee E, Caudill SP, Scherer G, Scherer M, Pluym N, Doig MV, Newland K, Murphy SE, Caron NJ, Sander LC, Shimizu M, Yamazaki H, Kim S, Langman LJ, Pritchett JS, Sniegoski LT, Li Y, Blount BC, Pirkle JL. Collaborative Method Performance Study of the Measurement of Nicotine, Its Metabolites, and Total Nicotine Equivalents in Human Urine. Cancer Epidemiol Biomarkers Prev. 2018 Sep;27(9):1083-1090. doi: 10.1158/1055-9965.EPI-17-1127. Epub 2018 May 31.
PMID: 29853481, PMC6125195
2. 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.
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
91704
Variables
Export Variables| Variable Name | Variable ID | Variable Description | dbGaP Mapping |
|---|
Measure Name
Biomarker of exposure to nicotine-containing products
Release Date
May 3, 2024
Definition
Cotinine is a major metabolite of nicotine and is an indicator of exposure to nicotine from tobacco or other nicotine containing products.
Purpose
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.
Keywords
Biomarker of exposure to nicotine-containing products - urine, 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 |
Publications
There are no publications listed for this protocol.