Analytical and Bioanalytical Chemistry

, Volume 407, Issue 14, pp 4101–4109 | Cite as

Simultaneous determination of nicotine and its nine metabolites in rat blood utilizing microdialysis coupled with UPLC–tandem mass spectrometry for pharmacokinetic application

  • Jian MaoEmail author
  • Yan Xu
  • Binbin Lu
  • Junhui Liu
  • Guangfeng Hong
  • Qidong Zhang
  • Shihao Sun
  • Jianxun ZhangEmail author
Research Paper


To develop a simple and rapid method for the simultaneous determination of nicotine and its nine metabolites in rat blood, an in vivo microdialysis sampling technique coupled with ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) was established for quantitation and characterization of the pharmacokinetics of nicotine and its metabolites. Microdialysis probes were inserted into the jugular vein of Sprague Dawley rats, and dialysates were collected after nicotine (0.5 mg/kg, i.p.) administration. Target analytes and corresponding deuterated internal standards were separated on a hydrophilic interaction liquid chromatography column (HILIC BEH 2.1. × 150 mm, 1.7 μm) and detected by UPLC–MS/MS under multiple reaction monitoring mode. The limits of quantification for nicotine and its nine metabolites ranged from 0.039 to 0.46 ng/mL. Intra- and inter-day precision and accuracy were well within the predefined limits of acceptability (<11 %). Pharmacokinetic results showed that the mean half-lives of nicotine, cotinine, nornicotine, norcotinine, nicotine-N′-oxide, cotinine-N′-oxide, trans-3′-hydroxy-cotinine, nicotine-N-glucuronide, cotinine-N-glucuronide, and trans-3′-hydroxy-cotinine-O-glucuronide in rat plasma were 63, 291, 175, 440, 251, 451, 322, 341, 488, and 516 min, respectively. The blood concentration-time profiles of nicotine and its nine metabolites indicate that nicotine is rapidly consumed after the administration and subsequently cotinine is generated as the main metabolite; meanwhile, cotinine and other eight minor metabolites exhibit longer retention times in rat body.

Graphical Abstract

Flowchart summarizing the experimental procedure


Nicotine Metabolites Microdialysis UPLC–MS/MS Pharmacokinetics 



This study was supported by NSFC (Grant no. 21307163) and the Presidential Science and Technology Development Foundation of ZTRI, China (No. 412011CA0280).


  1. 1.
    Benowitz NL (1999) Prim Care 26:611–631CrossRefGoogle Scholar
  2. 2.
    Batra V, Patkar AA, Berrettini WH, Weinstein SP, Leone FT (2003) Chest 123:1730–1739CrossRefGoogle Scholar
  3. 3.
    Thorgeirsson TE, Geller F, Sulem P, Rafnar T, Wiste A, Magnusson KP, Manolescu A, Thorleifsson G, Stefansson H, Ingason A, Stacey SN, Bergthorsson JT, Thorlacius S, Gudmundsson J, Jonsson T, Jakobsdottir M, Saemundsdottir J, Olafsdottir O, Gudmundsson LJ, Bjornsdottir G, Kristjansson K, Skuladottir H, Isaksson HJ, Gudbjartsson T, Jones GT, Mueller T, Gottsater A, Flex A, Aben KK, de Vegt F, Mulders PF, Isla D, Vidal MJ, Asin L, Saez B, Murillo L, Blondal T, Kolbeinsson H, Stefansson JG, Hansdottir I, Runarsdottir V, Pola R, Lindblad B, van Rij AM, Dieplinger B, Haltmayer M, Mayordomo JI, Kiemeney LA, Matthiasson SE, Oskarsson H, Tyrfingsson T, Gudbjartsson DF, Gulcher JR, Jonsson S, Thorsteinsdottir U, Kong A, Stefansson K (2008) Nature 452:638–642CrossRefGoogle Scholar
  4. 4.
    Malaiyandi V, Sellers EM, Tyndale RF (2005) Clin Pharmacol Ther 77:145–158CrossRefGoogle Scholar
  5. 5.
    Yildiz D (2004) Toxicon 43:619–632CrossRefGoogle Scholar
  6. 6.
    Boswell C, Curvall M, Elswick JRK, Leyden D (2000) Biomarkers 5:341–354Google Scholar
  7. 7.
    Fan Z, Xie FW, Xia QL, Wang S, Ding L, Liu HM (2008) Chromatographia 68:623–627CrossRefGoogle Scholar
  8. 8.
    Piller M, Gilch G, Scherer G, Scherer M (2014) J Chromatogr B 951–952:7–15CrossRefGoogle Scholar
  9. 9.
    Heinrich-Ramm R, Wegner R, Garde AH, Baur X (2002) Int J Hyg Environ Health 205:493–499CrossRefGoogle Scholar
  10. 10.
    Wielkoszynski T, Tyrpien K, Szumska M (2009) J Pharm Biomed Anal 49:1256–1260CrossRefGoogle Scholar
  11. 11.
    Jaakkola MS, Ma J, Yang G, Chin MF, Benowitz NL, Ceraso M, Samet JM (2003) Prev Med 36:282–290CrossRefGoogle Scholar
  12. 12.
    Man CN, Gam LH, Ismail S, Lajis R, Awang R (2006) J Chromatogr B 844:322–327CrossRefGoogle Scholar
  13. 13.
    Ghosheh OA, Browne D, Rogers T, de Leon J, Dwoskin LP, Crooks PA (2000) J Pharm Biomed Anal 23:543–549CrossRefGoogle Scholar
  14. 14.
    Baidoo EE, Clench MR, Smith RF, Tetler LW (2003) J Chromatogr B 796:303–313CrossRefGoogle Scholar
  15. 15.
    Shakleya DM, Huestis MA (2009) Anal Bioanal Chem 395:2349–2357CrossRefGoogle Scholar
  16. 16.
    Gray TR, Shakleya DM, Huestis MA (2009) Anal Bioanal Chem 393:1977–1990CrossRefGoogle Scholar
  17. 17.
    Concheiro M, Gray TR, Shakleya DM, Huestis MA (2010) Anal Bioanal Chem 398:915–924CrossRefGoogle Scholar
  18. 18.
    Concheiro M, Shakleya DM, Huestis MA (2011) Anal Bioanal Chem 400:69–78CrossRefGoogle Scholar
  19. 19.
    Johansen MJ, Newman RA, Madden T (1997) Pharmacotherapy 17:464–481Google Scholar
  20. 20.
    Verbeeck RK (2000) Adv Drug Deliv Rev 45:217–228CrossRefGoogle Scholar
  21. 21.
    Tsai TH (2002) J Agric Food Chem 50:6669–6674CrossRefGoogle Scholar
  22. 22.
    Tsai TH, Chen YF, Chou CJ, Chen CF (2000) J Chromatogr A 870:221–226CrossRefGoogle Scholar
  23. 23.
    Chang YL, Tsai PL, Chou YC, Tien JH, Tsai TH (2005) J Chromatogr A 1088:152–157CrossRefGoogle Scholar
  24. 24.
    Vieira-Brock PL, Miller EI, Nielsen SM, Fleckenstein AE, Wilkins DG (2011) J Chromatogr B 879:3465–3474CrossRefGoogle Scholar
  25. 25.
    Lang HL, Wang S, Zhang QD, Zhao BB, Wang L, Cao BJ, Wang J, Mao J, Zhang JX (2013) Anal Bioanal Chem 405:2083–2089CrossRefGoogle Scholar
  26. 26.
    Dobrinas M, Choong E, Noetzli M, Cornuz J, Ansermot N, Eap CB (2011) J Chromatogr B 879:3574–3582CrossRefGoogle Scholar
  27. 27.
    Kyerematen GA, Taylor LH, deBethizy JD, Vesell ES (1988) Drug Metab Dispos 16:125–129Google Scholar
  28. 28.
    Ghosheh O, Dwoskin LP, Li WK, Crooks PA (1999) Drug Metab Dispos 27:1448–1455Google Scholar
  29. 29.
    Kyerematen GA, Vesell ES (1991) Drug Metab Rev 23:3–41CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Jian Mao
    • 1
    Email author
  • Yan Xu
    • 2
  • Binbin Lu
    • 1
  • Junhui Liu
    • 1
  • Guangfeng Hong
    • 1
  • Qidong Zhang
    • 1
  • Shihao Sun
    • 1
  • Jianxun Zhang
    • 1
    Email author
  1. 1.Zhengzhou Tobacco Research Institute of CNTCZhengzhouChina
  2. 2.School of Life ScienceBeijing Institute of TechnologyBeijingChina

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