Accurate verification of nicotine and tobacco product use or abstinence using a multi-biomarker approach

Abstract

Cigarette smokers are exposed to over 7,000 chemicals, including many toxic and carcinogenic agents. Alternative non-combustible nicotine products considerably reduce exposure to harmful constituents. Extensive research on biomarkers of potential harm is crucial for assessing the early health impacts of smoking and the reduction of harm after quitting or switching to alternative products. However, such clinical trials are constrained by poor verification of self-reported product use, as common procedures using exhaled carbon monoxide (eCO) and cotinine strip tests are limited by short detection periods and non-specificity, respectively. Therefore, product-use-specific biomarkers of exposure (BoEs) are needed for accurate biochemical verification in studies evaluating the health impact of new products, such as electronic cigarettes. We conducted a cross-sectional study in 180 participants, including current, former, and never-smokers, and applied several BoEs to verify their use behavior. Our multi-biomarker approach monitored exposure to nicotine, tobacco-specific nitrosamines, acrylonitrile and propylene glycol (PG), which were significantly elevated in the current smoker group except PG, identifying six non-compliant subjects (4 current, 1 former and 1 never-smoker). The proposed biomarker panel outperformed eCO and was able to distinguish distinct use patterns, such as vaping vs. smoking, thereby enhancing data accuracy. Moreover, biochemically determined exposure variables such as carboxyhemoglobin and cotinine showed stronger correlations with BoEs than self-reported cigarette consumption. Therefore, the suggested panel is particularly valuable for non-controlled studies, where reliance on self-report can bias outcomes. Implementing the proposed verification strategy can improve study validity and strengthen evidence on the health impacts of switching to alternative products.

Keywords

Biomarkers of exposure / biomarkers of compliance / biochemical verification / cutoff value / detection period / tobacco harm reduction

References

[1] World Health Organization (WHO). Tobacco. 2025. https://www.who.int/news-room/fact-sheets/detail/tobacco. (accessed 2026-02-11).

[2] Goniewicz, M. L.; Gawron, M.; Smith, D. M.; Peng, M.; Jacob, P. 3rd; Benowitz, N. L. Exposure to nicotine and selected toxicants in cigarette smokers who switched to electronic cigarettes: a longitudinal within-subjects observational study.Nicotine Tob Res2017;19:160-7 PMCID:PMC5234360

[3] Yayan J,Biancosino C.Comparative systematic review on the safety of e-cigarettes and conventional cigarettes.Food Chem Toxicol2024;185:114507

[4] Scherer G.Suitability of biomarkers of biological effects (BOBEs) for assessing the likelihood of reducing the tobacco related disease risk by new and innovative tobacco products: a literature review.Regul Toxicol Pharmacol2018;94:203-33

[5] Benowitz NL,Foulds J.Biochemical verification of tobacco use and abstinence: 2019 update.Nicotine Tob Res2020;22:1086-97 PMCID:PMC7882145

[6] Pluym N,Scherer G.The potential of new nicotine and tobacco products as tools for people who smoke to quit combustible cigarettes - a systematic review of common practices and guidance towards a robust study protocol to measure cessation efficacy.Harm Reduct J2024;21:130 PMCID:PMC11225172

[7] SRNT Subcommittee on Biochemical Verification. Biochemical verification of tobacco use and cessation.Nicotine Tob Res2002;4:149-59

[8] CORESTA Guides. No. 29 - Best practice in the application of biomarkers of exposure as compliance measures in long-term and epidemiological studies of new nicotine and tobacco products. 2024. https://www.coresta.org/best-practice-application-biomarkers-exposure-compliance-measures-long-term-and-epidemiological. (accessed 2026-02-11).

[9] Feng J,Bernert JT.Urinary nicotine metabolites and self-reported tobacco use among adults in the population assessment of tobacco and health (PATH) study, 2013-2014.Nicotine Tob Res2022;24:768-77 PMCID:PMC9116621

[10] Wang Z,Tscharke BJ.High throughput and sensitive quantitation of tobacco-specific alkaloids and nitrosamines in wastewater.Talanta2024;277:126401

[11] Goniewicz ML,Peng MW.Elimination kinetics of the tobacco-specific biomarker and lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol.Cancer Epidemiol Biomarkers Prev2009;18:3421-5 PMCID:PMC2804843

[12] Bhandari D,Zhu W,Blount BC.Optimal cutoff concentration of urinary cyanoethyl mercapturic acid for differentiating cigarette smokers from nonsmokers.Nicotine Tob Res2022;24:761-7 PMCID:PMC8967795

[13] Jakubowski M,Pielas G.2-Cyanoethylmercapturic acid (CEMA) in the urine as a possible indicator of exposure to acrylonitrile.Br J Ind Med1987;44:834-40 PMCID:PMC1007929

[14] Burkhardt T,Scherer G.1,2-Propylene glycol: a biomarker of exposure specific to e-cigarette consumption.Separations2021;8:180

[15] Hiler M,Wolf CE,Nanco CR.Are urine propylene glycol or vegetable glycerin markers of e-cigarette use or abstinence?.Tob Regul Sci2020;6:235-41 PMCID:PMC9273112

[16] Yu DK,Sawchuk RJ.Pharmacokinetics of propylene glycol in humans during multiple dosing regimens.J Pharm Sci1985;74:876-9

[17] International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH). ICH guideline M10 on bioanalytical method validation and study sample analysis. 2022. https://www.ema.europa.eu/en/documents/scientific-guideline/ich-guideline-m10-bioanalytical-method-validation-step-5_en.pdf. (accessed 2026-02-11)

[18] Food and Drug Administration (FDA). Bioanalytical method validation - Guidance for Industry. 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf. (accessed 2026-02-11)

[19] Scherer G,Pluym N.Assessment of nicotine delivery and uptake in users of various tobacco/nicotine products.Curr Res Toxicol2022;3:100067 PMCID:PMC8938277

[20] Scherer G,Mütze J,Pluym N.Assessment of the exposure to tobacco-specific nitrosamines and minor tobacco alkaloids in users of various tobacco/nicotine products.Chem Res Toxicol2022;35:684-93

[21] Pluym N,Scherer G.Analysis of 18 urinary mercapturic acids by two high-throughput multiplex-LC-MS/MS methods.Anal Bioanal Chem2015;407:5463-76

[22] Scherer G,Papadopoulou E.A correlation study applied to biomarkers of internal and effective dose for acrylonitrile and 4-aminobiphenyl in smokers.Biomarkers2014;19:291-301 PMCID:PMC4133968

[23] Jaffé M.On the precipitation produced by picric acid in normal urine and on a new reaction of creatinine (in Germany). 1886.

[24] Benowitz NL,Jain S.Comparison of urine 4-(methylnitrosamino)-1-(3)pyridyl-1-butanol and cotinine for assessment of active and passive smoke exposure in urban adolescents.Cancer Epidemiol Biomarkers Prev2018;27:254-61 PMCID:PMC5835192

[25] Bendik PB,Pine BN.Anabasine and anatabine exposure attributable to cigarette smoking: National Health and Nutrition Examination Survey (NHANES) 2013-2014.Int J Environ Res Public Health2022;19:9744 PMCID:PMC9368097

[26] Tavares R,Monteiro M.Monitoring of exposure to acrylonitrile by determination of N-(2-cyanoethyl)valine at the N-terminal position of haemoglobin.Carcinogenesis1996;17:2655-60

[27] Maga M,Wachsmann A.Influence of air pollution on exhaled carbon monoxide levels in smokers and non-smokers. A prospective cross-sectional study.Environ Res2017;152:496-502

[28] Castleden CM.Carboxyhaemoglobin levels of smokers and non-smokers working in the City of London.Br J Ind Med1975;32:115-8 PMCID:PMC1008037

[29] Benowitz NL,Jacob P.Nicotine chemistry, metabolism, kinetics and biomarkers. In Nicotine psychopharmacology. Henningfield, J. E.; London, E. D.; Pogun, S.; Eds; Berlin: Springer Berlin Heidelberg, 2009; pp. 29-60. PMCID:PMC2953858

[30] Campo L,Bechtold P.Determinants of active and environmental exposure to tobacco smoke and upper reference value of urinary cotinine in not exposed individuals.Environ Res2016;148:154-63

[31] Edwards KC,Stanton CA.Urinary cotinine and cotinine + trans-3'-hydroxycotinine (TNE-2) cut-points for distinguishing tobacco use from nonuse in the United States: PATH study (2013-2014).Cancer Epidemiol Biomarkers Prev2021;30:1175-84 PMCID:PMC10337127

[32] Kim S.Overview of cotinine cutoff values for smoking status classification.Int J Environ Res Public Health2016;13:1236 PMCID:PMC5201377

[33] Paci E,Bauleo L,Forastiere F.Urinary cotinine concentration and self-reported smoking status in 1075 subjects living in central Italy.Int J Environ Res Public Health2018;15:804 PMCID:PMC5923846

[34] Park EY,Park E,Lee D.Optimum urine cotinine and NNAL levels to distinguish smokers from non-smokers by the changes in tobacco control policy in Korea from 2008 to 2018.Nicotine Tob Res2022;24:1821-8

[35] Piller M,Scherer G.Simple, fast and sensitive LC-MS/MS analysis for the simultaneous quantification of nicotine and 10 of its major metabolites.J Chromatogr B Analyt Technol Biomed Life Sci2014;951-2:7-15

[36] CORESTA Reports. Meta-analysis study to establish baseline levels of COHb and NEQs in smokers and non-smokers. 2019. https://www.coresta.org/meta-analysis-study-establish-baseline-levels-cohb-and-neqs-smokers-and-non-smokers-33007.html. (accessed 2026-02-11)

[37] Jacob, P. 3rd; Yu, L.; Shulgin, A. T.; Benowitz, N. L. Minor tobacco alkaloids as biomarkers for tobacco use: comparison of users of cigarettes, smokeless tobacco, cigars, and pipes.Am J Public Health1999;89:731-6 PMCID:PMC1508721

[38] Xia B,Guillot T.Tobacco-specific nitrosamines (NNAL, NNN, NAT, and NAB) exposures in the US Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).Nicotine Tob Res2021;23:573-83 PMCID:PMC7885786

[39] Colsoul ML,Onorati S.Novel proposed cutoff values for anatabine and anabasine in differentiating smokers from non-smokers.Clin Biochem2023;116:128-31

[40] Suh-Lailam BB,Carlisle HJ,McMillin GA.Reference interval determination for anabasine: a biomarker of active tobacco use.J Anal Toxicol2014;38:416-20

[41] Luo X,Chen M.Urinary cyanoethyl mercapturic acid, a biomarker of the smoke toxicant acrylonitrile, clearly distinguishes smokers from nonsmokers.Nicotine Tob Res2020;22:1744-7 PMCID:PMC7542649

[42] Camacho OM,Lowe F.Statistical analysis plan for “A randomised, controlled study to evaluate the effects of switching from cigarette smoking to using a tobacco heating product on health effect indicators in healthy subjects”.Contemp Clin Trials Commun2020;17:100535 PMCID:PMC7013164

[43] Scherer G.Carboxyhemoglobin and thiocyanate as biomarkers of exposure to carbon monoxide and hydrogen cyanide in tobacco smoke.Exp Toxicol Pathol2006;58:101-24

[44] Sánchez Fernández C,Zichen J.Correlation between blood carboxyhemoglobin levels and smoking.Eur Respir J2022;60:3618

[45] Whincup P,Lennon L.Carboxyhaemoglobin levels and their determinants in older British men.BMC Public Health2006;6:189 PMCID:PMC1555590

[46] Pojer R,Poulos V,Richmond R.Carboxyhemoglobin, cotinine, and thiocyanate assay compared for distinguishing smokers from non-smokers.Clin Chem1984;30:1377-80

[47] Ernst A.Carbon monoxide poisoning.N Engl J Med1998;339:1603-8

[48] Wang Y,Meng L.Urinary concentrations of monohydroxylated polycyclic aromatic hydrocarbons in adults from the U.S. Population Assessment of Tobacco and Health (PATH) Study Wave 1 (2013-2014).Environ Int2019;123:201-8 PMCID:PMC6331224

[49] Li Z,Bartell S.Excretion profiles and half-lives of ten urinary polycyclic aromatic hydrocarbon metabolites after dietary exposure.Chem Res Toxicol2012;25:1452-61 PMCID:PMC4618384

[50] Kavvadias D,Cheung F,Shepperd J.Determination of tobacco-specific N-nitrosamines in urine of smokers and non-smokers.Biomarkers2009;14:547-53