Method(s): LC-MS/MS (1).

What is measured on the same platform, click here.
Platform D: PLP, PL, PA, (PAr), PN, Thi, TMP, Ribo, FMN, Cysta, Neopt, Trp, Kyn, (KTR), KA, AA, HK, XA, (HK/XA), HAA, Pic, QA, NA, NAM, mNAM, Cot, OHCot, Trig

What is trans-3'-hydroxycotinine?

Trans-3'-hydroxycotinine (OHCot) is the main metabolite of cotinine with a half-life of 6.6 h. The metabolism of cotinine to OHCot is mediated by the enzyme cytochrome p450 2A6 (CYP2A6), encoded by the highly polymorphic CYP2A6 gene, with genotypes strongly associated with nicotine clearance and the nicotine metabolite ratio (NMR), defined as OHCot:cotinine (2). Other factors, including ethnicity, sex, hormones, smoking intensity, mentholated cigarettes, alcohol use, BMI, are weak predictors of NMR, and account for less than 8 % of NMR variation (3). Thus, NMR, OHCot/cotinine ratio, is a marker of CYP2A6 activity, and individuals can be categorized into “slow” versus “normal/fast metabolizers” based on their NMR value. The status of “slow metabolizer” has been associated with less nicotine dependence, lower smoking intensity, higher rates of smoking cessation and lower risk of lung cancer (2).

Performance of the assay

Lower limit of detection (LOD): 2 nmol/L.
Within-day CV: 3 %; between-day CV: 3 %.


Assessment of smoking status and tobacco exposure. Guiding smoking cessation pharmacotherapy.
The NMR, OHCot/cotinine ratio, is a marker of CYP2A6 activity. Based on NMR, individuals are categorized as “slow” versus “normal/fast metabolizers”.

Specimen, collection and processing

Matrix: Serum, EDTA plasma and urine
Volume: Minimum volume is 60 µL, but 200 µL is optimal and allows reanalysis.
Preparation and stability: The literature on stability of OHCot is inconsistent.

Transportation; for general instruction on transportation, click here.

Frozen, on dry ice.

Reported values, interpretation

The information below applies to measurement in serum or plasma.
The OHCot/cotinine ratio (NMR) varies considerably, in the range of 0.02 - 0.90. Smokers are classified as as slow (NMR < 0.31) or normal (NMR ≥ 0.31) metabolizers.
NMR is determined by CYP2A6 genotype. Other (weak) determinants are no source of misclassification. Nicotine patches and snuff give very high circulating concentrations of both cotinine and OHCot.
Intraclass correlation coefficient (ICC): 0.85 (4).


1. Midttun, O., Hustad, S., and Ueland, P.M. (2009). Quantitative profiling of biomarkers related to B-vitamin status, tryptophan metabolism and inflammation in human plasma by liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Sp 23, 1371-79.
2. Tanner, J. A., and Tyndale, R. F. (2017) Variation in CYP2A6 activity and personalized medicine. Journal of personalized medicine 7, E18.
3. Chenoweth, M.J., Novalen, M., Hawk, L.W., Schnoll, R.A., George, T.P., Cinciripini, P.M., Lerman, C., and Tyndale, R.F. (2014). Known and novel sources of variability in the nicotine metabolite ratio in a large sample of treatment-seeking smokers. Cancer Epidemiol Biomarkers Prev 23, 1773-782.
4. Hamilton, D.A., Mahoney, M.C., Novalen, M., Chenoweth, M.J., Heitjan, D.F., Lerman, C., Tyndale, R.F., and Hawk, L.W. (2015). Test-retest reliability and stability of the nicotine metabolite ratio among treatment-seeking smokers. Nicotine Tob Res 17, 1505-09.