Analytical and Bioanalytical Chemistry

, Volume 411, Issue 3, pp 669–678 | Cite as

Quantification of DEET and neonicotinoid pesticide biomarkers in human urine by online solid-phase extraction high-performance liquid chromatography-tandem mass spectrometry

  • Samuel E. Baker
  • Amanda Bishop Serafim
  • Pilar Morales-Agudelo
  • Meghan Vidal
  • Antonia M. Calafat
  • Maria OspinaEmail author
Research Paper


Neonicotinoid insecticides are widely used replacements for organophosphate and carbamate insecticides, but the extent of human exposure is largely unknown. On the other hand, based on urinary concentrations of DEET metabolites, human exposure to N,N-diethyl-m-toluamide (DEET) appears to be widespread. We developed a fast online solid-phase extraction high-performance liquid chromatography-isotope dilution tandem mass spectrometry (HPLC-MS/MS) method to measure in 200 μL of human urine the concentrations of six neonicotinoid biomarkers (acetamiprid, N-desmethyl-acetamiprid, clothianidin, imidacloprid, 5-hydroxy-imidacloprid, thiacloprid), and two DEET biomarkers (3-diethyl-carbamoyl benzoic acid, 3-ethyl-carbamoyl benzoic acid). Limits of detection ranged from 0.01 to 0.1 μg/L, depending on the biomarker. Accuracy ranged from 91 to 116% and precision ranged from 3.7 to 10 %RSD. The presented method can be used to increase our understanding of exposure to neonicotinoid insecticides and DEET, and to evaluate the potential health effects from such exposures.


DEET N,N-Diethyl-m-toluamide Neonicotinoids Biomarkers HPLC-MS/MS Online SPE 



We thank Charlie Chambers for technical assistance and Dr. Peter Kuklenyik for the diagram in the supplemental information.

Compliance with ethical standards

Conflict of interest

The authors declare they have no competing interests.

Supplementary material

216_2018_1481_MOESM1_ESM.pdf (328 kb)
ESM 1 (PDF 328 kb)


  1. 1.
    Douglas MR, Tooker JF. Large-scale deployment of seed treatments has driven rapid increase in use of neonicotinoid insecticides and preemptive pest min U.S. field crops. Environ Sci Technol. 2015;49(8):5088–97. Scholar
  2. 2.
    Goulson D. REVIEW: An overview of the environmental risks posed by neonicotinoid insecticides. J Appl Ecol. 2013;50(4):977–87. Scholar
  3. 3.
    Jeschke P, Nauen R, Schindler M, Elbert A. Overview of the status and global strategy for neonicotinoids. J Agric Food Chem. 2011;59(7):2897–908. Scholar
  4. 4.
    Simon-Delso N, Amaral-Rogers V, Belzunces LP, Bonmatin JM, Chagnon M, Downs C, et al. Systemic insecticides (neonicotinoids and fipronil): trends, uses, mode of action and metabolites. Environ Sci Pollut Res Int. 2015;22(1):5–34. Scholar
  5. 5.
    Tomizawa M, Casida JE. Neonicotinoid insecticide toxicology: mechanisms of selective action. Annu Rev Pharmacol Toxicol. 2005;45(1):247–68. Scholar
  6. 6.
    Bass C, Denholm I, Williamson MS, Nauen R. The global status of insect resistance to neonicotinoid insecticides. Pestic Biochem Physiol. 2015;121:78–87. Scholar
  7. 7.
    Matsuda K, Buckingham SD, Kleier D, Rauh JJ, Grauso M, Sattelle DB. Neonicotinoids: insecticides acting on insect nicotinic acetylcholine receptors. Trends Pharmacol Sci. 2001;22(11):573–80. Scholar
  8. 8.
    Kasiotis KM, Machera K. Neonicotinoids and their metabolites in human biomonitoring: a review. Hellenic Plant Protect J. 2015;8(2):33–45. Scholar
  9. 9.
    Casida JE. Neonicotinoid metabolism: compounds, substituents, pathways, enzymes, organisms, and relevance. J Agric Food Chem. 2011;59(7):2923–31. Scholar
  10. 10.
    Chen M, Tao L, McLean J, Lu C. Quantitative analysis of neonicotinoid insecticide residues in foods: implication for dietary exposures. J Agric Food Chem. 2014;62(26):6082–90. Scholar
  11. 11.
    Hladik ML, Main AR, Goulson D. Environmental risks and challenges associated with neonicotinoid insecticides. Environ Sci Technol. 2018.
  12. 12.
    Cimino AM, Boyles AL, Thayer KA, Perry MJ. Effects of neonicotinoid pesticide exposure on human health: a systematic review. Environ Health Perspect. 2017;125(2):155–62. Scholar
  13. 13.
    vanEngelsdorp D, Evans JD, Saegerman C, Mullin C, Haubruge E, Nguyen BK, et al. Colony collapse disorder: a descriptive study. PLoS One. 2009;4(8):e6481. Scholar
  14. 14.
    Hladik ML, Kolpin DW, Kuivila KM. Widespread occurrence of neonicotinoid insecticides in streams in a high corn and soybean producing region, USA. Environ Pollut. 2014;193:189–96. Scholar
  15. 15.
    Hallmann CA, Foppen RPB, van Turnhout CAM, de Kroon H, Jongejans E. Declines in insectivorous birds are associated with high neonicotinoid concentrations. Nature. 2014;511:341. Accessed 13 Aug 2013
  16. 16.
    Han W, Tian Y, Shen X. Human exposure to neonicotinoid insecticides and the evaluation of their potential toxicity: an overview. Chemosphere. 2018;192:59–65. Scholar
  17. 17.
    Dick RA, Kanne DB, Casida JE. Substrate specificity of rabbit aldehyde oxidase for nitroguanidine and nitromethylene neonicotinoid insecticides. Chem Res Toxicol. 2006;19(1):38–43. Scholar
  18. 18.
    Shi X, Dick RA, Ford KA, Casida JE. Enzymes and inhibitors in neonicotinoid insecticide metabolism. J Agric Food Chem. 2009;57(11):4861–6. Scholar
  19. 19.
    Schulz-Jander DA, Casida JE. Imidacloprid insecticide metabolism: human cytochrome P450 isozymes differ in selectivity for imidazolidine oxidation versus nitroimine reduction. Toxicol Lett. 2002;132(1):65–70. Scholar
  20. 20.
    Ford KA, Casida JE. Chloropyridinyl neonicotinoid insecticides: diverse molecular substituents contribute to facile metabolism in mice. Chem Res Toxicol. 2006;19(7):944–51. Scholar
  21. 21.
    Ford KA, Casida JE. Unique and common metabolites of thiamethoxam, clothianidin, and dinotefuran in mice. Chem Res Toxicol. 2006;19(11):1549–56. Scholar
  22. 22.
    Marfo JT, Fujioka K, Ikenaka Y, Nakayama SM, Mizukawa H, Aoyama Y, et al. Relationship between urinary N-desmethyl-acetamiprid and typical symptoms including neurological findings: a prevalence case-control study. PLoS One. 2015;10(11):e0142172. Scholar
  23. 23.
    Taira K, Fujioka K, Aoyama Y. Qualitative profiling and quantification of neonicotinoid metabolites in human urine by liquid chromatography coupled with mass spectrometry. PLoS One. 2013;8(11):e80332. Scholar
  24. 24.
    Nomura H, Ueyama J, Kondo T, Saito I, Murata K, Iwata T, et al. Quantitation of neonicotinoid metabolites in human urine using GC-MS. J Chromatogr B. 2013;941:109–15. Scholar
  25. 25.
    Uroz FJ, Arrebola FJ, Egea-Gonzalez FJ, Martinez-Vidal JL. Monitoring of 6-chloronicotinic acid in human urine by gas chromatography-tandem mass spectrometry as indicator of exposure to the pesticide imidacloprid. Analyst. 2001;126(8):1355–8.Google Scholar
  26. 26.
    Harada KH, Tanaka K, Sakamoto H, Imanaka M, Niisoe T, Hitomi T, et al. Biological monitoring of human exposure to neonicotinoids using urine samples, and neonicotinoid excretion kinetics. PLoS One. 2016;11(1):e0146335. Scholar
  27. 27.
    Ueyama J, Nomura H, Kondo T, Saito I, Ito Y, Osaka A, et al. Biological monitoring method for urinary neonicotinoid insecticides using LC-MS/MS and its application to Japanese adults. J Occup Health. 2014;56(6):461–8. Scholar
  28. 28.
    Wang L, Liu T, Liu F, Zhang J, Wu Y, Sun H. Occurrence and profile characteristics of the pesticide imidacloprid, preservative parabens, and their metabolites in human urine from rural and urban China. Environ Sci Technol. 2015;49(24):14633–40. Scholar
  29. 29.
    Yamamuro T, Ohta H, Aoyama M, Watanabe D. Simultaneous determination of neonicotinoid insecticides in human serum and urine using diatomaceous earth-assisted extraction and liquid chromatography–tandem mass spectrometry. J Chromatogr B. 2014;969:85–94. Scholar
  30. 30.
    Zhang Q, Li Z, Chang CH, Lou JL, Zhao MR, Lu C. Potential human exposures to neonicotinoid insecticides: a review. Environ Pollut. 2018;236:71–81. Scholar
  31. 31.
    López-García M, Romero-González R, Lacasaña M, Garrido Frenich A. Semiautomated determination of neonicotinoids and characteristic metabolite in urine samples using TurboFlow™ coupled to ultra high performance liquid chromatography coupled to Orbitrap analyzer. J Pharm Biomed Anal. 2017;146:378–86. Scholar
  32. 32.
    CDC (2017) Protection against mosquitoes, ticks, & other Arthropods, Centers for Disease Control and Prevention National Center for Emerging and Zoonotic Infectious Diseases Accessed 5 July 2018.
  33. 33.
    EPA (2017) DEET. Accessed 5 July 2018.
  34. 34.
    EPA (2017) Find the insect repellent that is right for you. Accessed 5 July 2018.
  35. 35.
    Calafat AM, Baker SE, Wong LY, Bishop AM, Morales AP, Valentin-Blasini L. Novel exposure biomarkers of N,N-diethyl-m-toluamide (DEET): data from the 2007-2010 National Health and Nutrition Examination Survey. Environ Int. 2016;92-93:398–404. Scholar
  36. 36.
    ATSDR (2017) Toxicological profile for DEET (N,N-diethyl-meta-toluamide). U.S. Department of Health and Human Services. Agency for Toxic Substances and Disease Registry. Accessed 13 Aug 2018
  37. 37.
    Caudill SP, Schleicher RL, Pirkle JL. Multi-rule quality control for the age-related eye disease study. Stat Med. 2008;27(20):4094–106. Scholar
  38. 38.
    FDA (2018) Bionalytical method validation-guidance for industry. May 2018 edn. Food and Drug Administration Center for Drug Evaluation and Research,Google Scholar
  39. 39.
    Bader MBD, Gæen T, Schaller KH, Scherer G, Angerer J. Reliability criteria for analytical methods [Biomonitoring Methods, 2010]. In: GmbH&Co W-VV, editor. The MAK-Collection for occupational health and safety, vol 12, Biomonitoring Methods., vol Part IV; 2010. Scholar
  40. 40.
    Taylor J. Quality assurance of chemical measurements. 1st ed: CRC Press; 1987.Google Scholar
  41. 41.
    Matuszewski BK. Standard line slopes as a measure of a relative matrix effect in quantitative HPLC-MS bioanalysis. J Chromatogr B Analyt Technol Biomed Life Sci. 2006;830(2):293–300. Scholar
  42. 42.
    Kuklenyik P, Baker SE, Bishop AM, Morales AP, Calafat AM. On-line solid phase extraction-high performance liquid chromatography-isotope dilution-tandem mass spectrometry approach to quantify N,N-diethyl-m-toluamide and oxidative metabolites in urine. Anal Chim Acta. 2013;787:267–73. Scholar
  43. 43.
    Osaka A, Ueyama J, Kondo T, Nomura H, Sugiura Y, Saito I, et al. Exposure characterization of three major insecticide lines in urine of young children in Japan—neonicotinoids, organophosphates, and pyrethroids. Environ Res. 2016;147:89–96. Scholar
  44. 44.
    CDC (2018) Fourth National Report on Human Exposure to Environmental Chemicals, Updated Tables, March 2018 Centers for Disease Control and Prevention; National Center for Environmental Health; Division of Laboratory Sciences. . Accessed July 5, 2018.

Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply 2018

Authors and Affiliations

  • Samuel E. Baker
    • 1
  • Amanda Bishop Serafim
    • 1
  • Pilar Morales-Agudelo
    • 1
    • 2
  • Meghan Vidal
    • 1
    • 2
  • Antonia M. Calafat
    • 1
  • Maria Ospina
    • 1
    Email author
  1. 1.Division of Laboratory Sciences, National Center for Environmental HealthCenters for Disease Control and PreventionAtlantaUSA
  2. 2.Battelle Memorial InstituteAtlantaUSA

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