Protocol - Nicotine Metabolite Ratio - Serum and Saliva

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This is the laboratory protocol for measuring cotinine and trans-3’-hydroxycotinine in serum and saliva, which is used by the Centers for Disease Control and Prevention and for National Health and Nutrition Examination Survey cotinine analyses. The ratio of hydroxycotinine to cotinine, known as the nicotine metabolite ratio, is correlated with the rate of nicotine metabolism in smokers and may be used to select the optimal type of smoking cessation medication. 

Specific Instructions

Cotinine (COT) and trans-3’-hydroxycotinine (HC) can be measured in serum, plasma, urine, and saliva. COT concentrations tend to be three to eight times higher in urine than in serum; however, plasma or serum is the fluid of choice for studies requiring a quantitative assessment of exposure. For that reason, serum was chosen as the matrix for the National Health and Nutrition Examination Survey COT analyses. In serum, HC concentrations tend to be two to four times lower than COT concentrations. This same method is used to measure COT and HC in saliva by substituting suitable saliva quality control pools. All other aspects, including calibration, cleanup, and analysis, are identical to serum procedures.

Sample processing does not require anticoagulants, special preservatives, or unusual sterility procedures. However, the Working Group notes that plasma from anticoagulated blood can also be used. Blood can be collected from venipuncture by using standard equipment. Allow the blood to clot for a minimum of 30 minutes and up to 2 hours to create maximum serum yield. Transfer the serum to polypropylene cryogenic, screw-cap vials and freeze. Collection of saliva is most conveniently accomplished using a Salivette® or similar commercial device. Salivettes may be frozen directly after sample collection for subsequent transfer to the laboratory without any further processing required. The testing laboratory should be contacted before samples are collected to confirm the suitability of any equipment used to collect, process, or store samples intended for these analyses.

Nicotine metabolite ratio (NMR) data are commonly analyzed by quartiles, based on data for the particular study population. Typical NMR ranges and distributions vary by race and sex, and reference range information should be obtained as part of the individual research study being conducted.

The Smoking Cessation, Harm Reduction, and Biomarkers Working Group recommends that the protocol can be used for adults aged 18 years or older who can provide a blood or saliva sample.


Cotinine (COT) and trans-3’-hydroxycotinine (HC) are measured by an isotope-dilution high-performance liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometric (ID HPLC-APCI MS/MS) method. Briefly, the serum sample is spiked with methyl-D3-COT and methyl-D3-HC as internal standards. The sample is basified and then applied to a supported liquid extraction (SLE) plate. The analytes are extracted with an isopropanol/methylene chloride mixture, the organic extract is concentrated, and the residue is injected onto a C18 HPLC column. The eluent from these injections is monitored by APCI-MS/MS. The m/z 80 product ion from the m/z 177 quasi-molecular ion is measured for COT and the m/z 80 product ion from the m/z 193 quasi-molecular ion is measured for HC. Additional ions for the internal standards and for confirmation are also monitored for the respective compounds. Analyte concentrations are derived from the area ratios of native-to-labeled compounds in the sample by comparisons to a standard curve. The limit of detection (LOD) is 15 pg/ml.

The nicotine metabolite ratio is then calculated as the ratio of HC to COT.



Personnel and Training Required

Laboratory training for sample preparation, sample handling, and in the use of liquid chromatography and tandem mass spectrometry is required.

Equipment Needs

This method requires high-performance liquid chromatography and tandem mass spectrometry.

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






Selection Rationale

Cotinine (COT) and trans-3’-hydroxycotinine (HC) are the primary metabolites of nicotine. The concentration of COT in body fluids can be used alone or in tandem with HC as a marker for active smoking. Because their concentrations are greater and their elimination half-lives significantly longer, these metabolites are generally preferred over nicotine itself as biomarkers. 

The ratio of HC to COT is called the nicotine metabolite ratio (NMR). It is highly correlated with the rate of nicotine metabolism in smokers. It is believed that the severity of nicotine dependence is related to an individual's rate of nicotine metabolism—the higher the NMR, the faster the metabolism of nicotine and the more dependent on nicotine the individual is. The conversion of nicotine to COT, as well as the conversion of COT to HC, is largely mediated by the liver enzyme cytochrome P450 2A6 (CYP2A6). Thus, the NMR provides a convenient measure to phenotype individuals for CYP2A6 activity. CYP2A6 is also responsible for metabolic activation of carcinogenic tobacco-specific nitrosamines. Therefore, the NMR can be helpful in the development of individual pharmacotherapies for nicotine dependence (Centers for Disease Control and Prevention, 2018).



Human Phenotype Ontology Plasma/serum xenobiotic HP:0500100 HPO
Derived Variables


Process and Review

Not Applicable

Protocol Name from Source

National Health and Nutrition Examination Survey (NHANES) Cotinine and Hydroxycotinine Analysis


Centers for Disease Control and Prevention (CDC), National Center for Health Statistics (NCHS). (2018). Laboratory procedure manual for cotinine and hydroxycotinine in serum and saliva. Hyattsville, MD: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention.

General References

Carroll, D. M., Murphy, S. E., Benowitz, N. L., Strasser, A. A., Kotlyar, M., Hecht, S. S., Carmella, S. G., McClernon, F. J., Pacek, L. R., Dermody, S. S., Vandrey, R. G., Donny, E. C., & Hatsukami, D. K. (2020). Relationships between the nicotine metabolite ratio and a panel of exposure and effect biomarkers: Findings from two studies of U.S. commercial cigarette smokers. Cancer Epidemiology, Biomarkers & Prevention, 29(4), 871-879.

Dempsey, D., Tutka, P., Jacob, P. III, Allen, F., Schoedel, K., Tyndale, R. F., & Benowitz, N. L. (2004). Nicotine metabolite ratio as an index of cytochrome P450 2A6 metabolic activity. Clinical Pharmacology and Therapeutics, 76(1), 64-72.

Lerman, C., Schnoll, R. A., Hawk, L. W., Jr., Cinciripini, P., George, T. P., Wileyto, E. P., Swan, G. E., Benowitz, N. L., Heitjan, D. F., Tyndale, R. F., & PGRN-PNAT Research Group. (2015). Use of the nicotine metabolite ratio as a genetically informed biomarker of response to nicotine patch or varenicline for smoking cessation: A randomized, double-blind placebo-controlled trial. The Lancet. Respiratory Medicine, 3(2), 131-138.

Park, S. L., Tiirikainen, M. I., Patel, Y. M., Wilkens, L. R., Stram, D. O., Le Marchand, L., & Murphy, S. E. (2016). Genetic determinants of CYP2A6 activity across racial/ethnic groups with different risks of lung cancer and effect on their smoking intensity. Carcinogenesis, 37(3), 269-279.

St. Helen, G., Novalen, M., Heitjan, D. F., Dempsey, D., Jacob, P. III, Aziziyeh, A., Wing, V. C., George, T. P., Tyndale, R. F., & Benowitz, N. L. (2012). Reproducibility of the nicotine metabolite ratio in cigarette smokers. Cancer Epidemiology, Biomarkers & Prevention, 21(7), 1105-1114.

Protocol ID


Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX330401010000 Concentration of cotinine (COT) N/A
PX330401020000 Concentration of trans-3'-hydroxycotinine (HC) N/A
Smoking Cessation, Harm Reduction and Biomarkers
Measure Name

Nicotine Metabolite Ratio - Serum and Saliva

Release Date

February 23, 2021


The ratio of nicotine metabolites trans-3’ hydroxycotinine and cotinine, a biomarker of CYP2A6 enzyme activity.


To measure the activity of the enzyme CYP2A6, which is the primary enzyme responsible for nicotine metabolism. A genetically informed biomarker of nicotine clearance, the nicotine metabolite ratio is associated with smoking cessation success and treatment response in clinical trials.


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Measure Protocols
Protocol ID Protocol Name
330401 Nicotine Metabolite Ratio - Serum and Saliva

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