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Isotope dilution-LC-MS/MS method for quantification of the urinary cotinine-to-creatinine ratio

  • Katharina Habler ORCID logo EMAIL logo , Michael Paal and Michael Vogeser
Published/Copyright: March 25, 2020
Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Clinical Chemistry and Laboratory Medicine (CCLM) Volume 58 Issue 9

Abstract

Background

Appropriate monitoring of tobacco smoking is extremely important in several areas of medicine, e.g. management of chronic obstructive pulmonary disease (COPD), epidemiological surveys, and allocation of heart or lung transplants. The major metabolite of nicotine is cotinine that is increasingly used as a laboratory parameter for assessing tobacco smoke exposure.

Methods

Creatinine and cotinine were analyzed simultaneously in urine by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in one run within 3 min using a biphenyl column. For quantification, the respective stable-isotope-labeled standards were used.

Results

Detuning and measuring a natural isotope of creatinine as precursor and product ion allowed a simultaneous quantification of creatinine and cotinine. The method revealed robust validation results. For both analytes, inaccuracy and imprecision of the quality control and external quality assessment (EQA) samples were ≤−11.1%.

Conclusions

One essential novelty of the method presented here is the simultaneous quantification of creatinine and cotinine covered by one analytical method. Despite the very different natural concentrations of creatinine and cotinine, this allows the immediate reporting of the cotinine-to-creatinine ratio without the need for a separate creatinine analysis.

Keywords: cotinine; creatinine; tobacco smoking exposure; UHPLC-MS/MS; urine
Corresponding author: Dr. rer. nat. Katharina Habler, Institute of Laboratory Medicine, Hospital of the University of Munich (LMU), Marchioninistr. 15, 81377 Munich, Germany, Phone: +49 89 4400 76248

  1. Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Ahluwalia IB, Smith T, Arrazola RA, Palipudi KM, Garcia de Quevedo I, Prasad VM, et al. Current tobacco smoking, quit attempts, and knowledge about smoking risks among persons aged >/=15 years – Global Adult Tobacco Survey, 28 countries, 2008–2016. MMWR Morb Mortal Wkly Rep 2018;67:1072–6.10.15585/mmwr.mm6738a7Search in Google Scholar

2. GBD 2015 Tobacco Collaborators. Smoking prevalence and attributable disease burden in 195 countries and territories, 1990–2015: a systematic analysis from the Global Burden of Disease Study 2015. Lancet 2017;389:1885–906.10.1016/S0140-6736(17)30819-XSearch in Google Scholar

3. Fagerstrom K. The epidemiology of smoking: health consequences and benefits of cessation. Drugs 2002;62(Suppl 2):1–9.10.2165/00003495-200262002-00001Search in Google Scholar

4. GBD 2017 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 354 diseases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017. Lancet 2018;392:1789–858.10.1016/S0140-6736(18)32279-7Search in Google Scholar

5. Glanville AR, Estenne M. Indications, patient selection and timing of referral for lung transplantation. Eur Respir J 2003;22:845–52.10.1183/1025448x.00026001Search in Google Scholar

6. Richtlinie gemäß § 16 Abs. 1 S. 1 Nrn. 2 u. 5 TPG für die Wartelistenführung und Organvermittlung zur Lungentransplantation. Dtsch Arztebl International 2017;114:1948-.Search in Google Scholar

7. Corbett C, Armstrong MJ, Neuberger J. Tobacco smoking and solid organ transplantation. Transplantation 2012;94:979–87.10.1097/TP.0b013e318263ad5bSearch in Google Scholar PubMed

8. Wells AJ, English PB, Posner SF, Wagenknecht LE, Perez-Stable EJ. Misclassification rates for current smokers misclassified as nonsmokers. Am J Public Health 1998;88:1503–9.10.2105/AJPH.88.10.1503Search in Google Scholar PubMed PubMed Central

9. Pichini S, Basagana XB, Pacifici R, Garcia O, Puig C, Vall O, et al. Cord serum cotinine as a biomarker of fetal exposure to cigarette smoke at the end of pregnancy. Environ Health Perspect 2000;108:1079–83.10.1289/ehp.001081079Search in Google Scholar PubMed PubMed Central

10. Mansi G, Raimondi F, Pichini S, Capasso L, Sarno M, Zuccaro P, et al. Neonatal urinary cotinine correlates with behavioral alterations in newborns prenatally exposed to tobacco smoke. Pediatr Res 2007;61:257–61.10.1203/pdr.0b013e31802d89ebSearch in Google Scholar PubMed

11. Pellegrini M, Rotolo MC, La Grutta S, Cibella F, Garcia-Algar O, Bacosi A, et al. Assessment of exposure to environmental tobacco smoke in young adolescents following implementation of smoke-free policy in Italy. Forensic Sci Int 2010;196:97–100.10.1016/j.forsciint.2009.12.041Search in Google Scholar PubMed

12. Benowitz NL, Jain S, Dempsey DA, Nardone N, Helen GS, Jacob 3rd P. Urine cotinine screening detects nearly ubiquitous tobacco smoke exposure in urban adolescents. Nicotine Tob Res 2017;19:1048–1054.10.1093/ntr/ntw390Search in Google Scholar

13. Benowitz NL, Kuyt F, Jacob 3rd P, Jones RT, Osman AL. Cotinine disposition and effects. Clin Pharmacol Ther 1983;34: 604–11.10.1038/clpt.1983.222Search in Google Scholar

14. Lewis DF, Dickins M, Lake BG, Eddershaw PJ, Tarbit MH, Goldfarb PS. Molecular modelling of the human cytochrome P450 isoform CYP2A6 and investigations of CYP2A substrate selectivity. Toxicology 1999;133:1–33.10.1016/S0300-483X(98)00149-8Search in Google Scholar

15. Donato MT, Viitala P, Rodriguez-Antona C, Lindfors A, Castell JV, Raunio H, et al. CYP2A5/CYP2A6 expression in mouse and human hepatocytes treated with various in vivo inducers. Drug Metab Dispos 2000;28:1321–6.Search in Google Scholar

16. Benowitz NL, Jacob 3rd P, Fong I, Gupta S. Nicotine metabolic profile in man: comparison of cigarette smoking and transdermal nicotine. J Pharmacol Exp Ther 1994;268: 296–303.Search in Google Scholar

17. Caldwell WS, Greene JM, Byrd GD, Chang KM, Uhrig MS, deBethizy JD, et al. Characterization of the glucuronide conjugate of cotinine: a previously unidentified major metabolite of nicotine in smokers’ urine. Chem Res Toxicol 1992;5:280–5.10.1021/tx00026a021Search in Google Scholar

18. Kuehl GE, Murphy SE. N-glucuronidation of trans-3’-hydroxycotinine by human liver microsomes. Chem Res Toxicol 2003;16:1502–6.10.1021/tx034173oSearch in Google Scholar

19. Benowitz NL. Cotinine as a biomarker of environmental tobacco smoke exposure. Epidemiol Rev 1996;18:188–204.10.1093/oxfordjournals.epirev.a017925Search in Google Scholar

20. Benowitz NL. Toxicity of nicotine: implications with regard to nicotine replacement therapy. Prog Clin Biol Res 1988;261: 187–217.Search in Google Scholar

21. Florescu A, Ferrence R, Einarson T, Selby P, Soldin O, Koren G. Methods for quantification of exposure to cigarette smoking and environmental tobacco smoke: focus on developmental toxicology. Ther Drug Monit 2009;31:14–30.10.1097/FTD.0b013e3181957a3bSearch in Google Scholar

22. Jacob 3rd P, Wilson M, Benowitz NL. Improved gas chromatographic method for the determination of nicotine and cotinine in biologic fluids. J Chromatogr 1981;222:61–70.10.1016/S0378-4347(00)81033-6Search in Google Scholar

23. Hariharan M, VanNoord T. Liquid-chromatographic determination of nicotine and cotinine in urine from passive smokers: comparison with gas chromatography with a nitrogen-specific detector. Clin Chem 1991;37:1276–80.10.1093/clinchem/37.7.1276Search in Google Scholar

24. Jacob 3rd P, Yu L, Wilson M, Benowitz NL. Selected ion monitoring method for determination of nicotine, cotinine and deuterium-labeled analogs: absence of an isotope effect in the clearance of (S)-nicotine-3’,3’-d2 in humans. Biol Mass Spectrom 1991;20:247–52.10.1002/bms.1200200503Search in Google Scholar PubMed

25. Xu X, Iba MM, Weisel CP. Simultaneous and sensitive measurement of anabasine, nicotine, and nicotine metabolites in human urine by liquid chromatography-tandem mass spectrometry. Clin Chem 2004;50:2323–30.10.1373/clinchem.2004.038489Search in Google Scholar

26. Miller EI, Norris HR, Rollins DE, Tiffany ST, Wilkins DG. A novel validated procedure for the determination of nicotine, eight nicotine metabolites and two minor tobacco alkaloids in human plasma or urine by solid-phase extraction coupled with liquid chromatography-electrospray ionization-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2010;878:725–37.10.1016/j.jchromb.2009.12.018Search in Google Scholar

27. Niedbala RS, Haley N, Kardos S, Kardos K. Automated homogeneous immunoassay analysis of cotinine in urine. J Anal Toxicol 2002;26:166–70.10.1093/jat/26.3.166Search in Google Scholar

28. Dunlop AJ, Clunie I, Stephen DW, Allison JJ. Determination of cotinine by LC-MS-MS with automated solid-phase extraction. J Chromatogr Sci 2014;52:351–6.10.1093/chromsci/bmt038Search in Google Scholar

29. Abdallah IA, Hammell DC, Stinchcomb AL, Hassan HE. A fully validated LC-MS/MS method for simultaneous determination of nicotine and its metabolite cotinine in human serum and its application to a pharmacokinetic study after using nicotine transdermal delivery systems with standard heat application in adult smokers. J Chromatogr B Analyt Technol Biomed Life Sci 2016;1020:67–77.10.1016/j.jchromb.2016.03.020Search in Google Scholar

30. Tretzel L, Thomas A, Piper T, Hedeland M, Geyer H, Schanzer W, et al. Fully automated determination of nicotine and its major metabolites in whole blood by means of a DBS online-SPE LC-HR-MS/MS approach for sports drug testing. J Pharm Biomed Anal 2016;123:132–40.10.1016/j.jpba.2016.02.009Search in Google Scholar

31. Jarvis M, Tunstall-Pedoe H, Feyerabend C, Vesey C, Salloojee Y. Biochemical markers of smoke absorption and self reported exposure to passive smoking. J Epidemiol Community Health 1984;38:335–9.10.1136/jech.38.4.335Search in Google Scholar

32. Thompson SG, Barlow RD, Wald NJ, Van Vunakis H. How should urinary cotinine concentrations be adjusted for urinary creatinine concentration? Clin Chim Acta 1990;187:289–95.10.1016/0009-8981(90)90114-8Search in Google Scholar

33. Luginbuhl M, Weinmann W. Creatinine in urine – a method comparison. Drug Test Anal 2017;9:1537–41.10.1002/dta.2166Search in Google Scholar PubMed

34. Chadwick CA, Keevil B. Measurement of cotinine in urine by liquid chromatography tandem mass spectrometry. Ann Clin Biochem 2007;44:455–62.10.1258/000456307781645996Search in Google Scholar PubMed

35. Wielders JP, Mink JK. Analysis of vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid in human urine by high-performance liquid chromatography and fluorometry. J Chromatogr 1984;310:379–85.10.1016/0378-4347(84)80103-6Search in Google Scholar

36. European Medicines Agency, Guideline on Bioanalytical Method Validation, London, UK, 2011. Accessed January 2020: https://www.ema.europa.eu/en/documents/ scientific-guideline/guideline-bioanalytical-method- validation_en.pdf.Search in Google Scholar

37. Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal Chem 2003;75:3019–30.10.1021/ac020361sSearch in Google Scholar

38. Bonfiglio R, King RC, Olah TV, Merkle K. The effects of sample preparation methods on the variability of the electrospray ionization response for model drug compounds. Rapid Commun Mass Spectrom 1999;13:1175–85.10.1002/(SICI)1097-0231(19990630)13:12<1175::AID-RCM639>3.0.CO;2-0Search in Google Scholar

39. Moyer TP, Charlson JR, Enger RJ, Dale LC, Ebbert JO, Schroeder DR, et al. Simultaneous analysis of nicotine, nicotine metabolites, and tobacco alkaloids in serum or urine by tandem mass spectrometry, with clinically relevant metabolic profiles. Clin Chem 2002;48:1460–71.10.1093/clinchem/48.9.1460Search in Google Scholar

40. Evangelista L, Ter-Galstanyan A, Moser DK, Dracup K. Smoking among women following heart transplantation: should we be concerned? Prog Cardiovasc Nurs 2009;24:119–23.10.1111/j.1751-7117.2009.00049.xSearch in Google Scholar

41. Ruttens D, Verleden SE, Goeminne PC, Poels K, Vandermeulen E, Godderis L, et al. Smoking resumption after lung transplantation: standardised screening and importance for long-term outcome. Eur Respir J 2014;43:300–3.10.1183/09031936.00141113Search in Google Scholar

42. Perez-Stable EJ, Benowitz NL, Marin G. Is serum cotinine a better measure of cigarette smoking than self-report? Prev Med 1995;24:171–9.10.1006/pmed.1995.1031Search in Google Scholar

43. Kunutsor SK, Spee JM, Kieneker LM, Gansevoort RT, Dullaart RP, Voerman AJ, et al. Self-reported smoking, urine cotinine, and risk of cardiovascular disease: findings from the PREVEND (Prevention of Renal and Vascular End-Stage Disease) Prospective Cohort Study. J Am Heart Assoc 2018;7:1–15.10.1161/JAHA.118.008726Search in Google Scholar

44. Kim S. Overview of cotinine cutoff values for smoking status classification. Int J Environ Res Public Health 2016;13:1–15.10.1016/B978-0-12-813035-3.00051-4Search in Google Scholar

45. Campo L, Polledri E, Bechtold P, Gatti G, Ranzi A, Lauriola P, et al. Determinants of active and environmental exposure to tobacco smoke and upper reference value of urinary cotinine in not exposed individuals. Environ Res 2016;148:154–63.10.1016/j.envres.2016.03.029Search in Google Scholar
Received: 2020-02-20
Accepted: 2020-02-28
Published Online: 2020-03-25
Published in Print: 2020-08-27

©2020 Walter de Gruyter GmbH, Berlin/Boston

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https://doi.org/10.1515/cclm-2020-0177
Keywords for this article
cotinine; creatinine; tobacco smoking exposure; UHPLC-MS/MS; urine

Articles in the same Issue

  1. Frontmatter
  2. Editorial
  3. Measurement uncertainty: light in the shadows
  4. Review
  5. Childhood obesity: an overview of laboratory medicine, exercise and microbiome
  6. Mini review
  7. The utility of measurement uncertainty in medical laboratories
  8. Opinion Paper
  9. Do genetic polymorphisms in angiotensin converting enzyme 2 (ACE2) gene play a role in coronavirus disease 2019 (COVID-19)?
  10. Opinion Papers
  11. The current status and future prospects of precision medicine
  12. Precision medicine in medical oncology: hope, disappointment and reality
  13. IFCC Papers
  14. Laboratory practices to mitigate biohazard risks during the COVID-19 outbreak: an IFCC global survey
  15. Operational considerations and challenges of biochemistry laboratories during the COVID-19 outbreak: an IFCC global survey
  16. Genetics and Molecular Diagnostics
  17. Diverse fragment lengths dismiss size selection for serum cell-free DNA: a comparative study of serum and plasma samples
  18. General Clinical Chemistry and Laboratory Medicine
  19. Quantification of plasma remdesivir and its metabolite GS-441524 using liquid chromatography coupled to tandem mass spectrometry. Application to a Covid-19 treated patient
  20. Isotope dilution-LC-MS/MS method for quantification of the urinary cotinine-to-creatinine ratio
  21. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based assay to profile 20 plasma steroids in endocrine disorders
  22. Assessment of antinuclear antibodies by indirect immunofluorescence assay: report from a survey by the American Association of Medical Laboratory Immunologists
  23. Evaluation of a novel extended automated particle-based multi-analyte assay for the detection of autoantibodies in the diagnosis of primary biliary cholangitis
  24. Reference Values and Biological Variations
  25. Continuous, complete and comparable NT-proBNP reference ranges in healthy children
  26. Reference-mean-centered statistical quality control
  27. Hematology and Coagulation
  28. Top-down and bottom-up approaches for the estimation of measurement uncertainty in coagulation assays
  29. Cancer Diagnostics
  30. Tumor-associated exosomal miRNA biomarkers to differentiate metastatic vs. nonmetastatic non-small cell lung cancer
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  32. Cardiovascular Diseases
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  34. The clinically effective use of cardiac markers by restructuring laboratory profiles at Cardiology wards
  35. Infectious Diseases
  36. Lower nasopharyngeal viral load during the latest phase of COVID-19 pandemic in a Northern Italy University Hospital
  37. SARS-CoV-2 RNA identification in nasopharyngeal swabs: issues in pre-analytics
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  50. The new era of LED microscopes in immunofluorescence anti-nuclear antibody (ANA) testing
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