Skip to main content

Advertisement

SpringerLink
Log in

Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam, meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Among the new psychoactive substances (NPS), so-called designer benzodiazepines have become of particular importance over the last 2 years, due to their increasing availability on the internet drug market. Therapeutically used nitrobenzodiazepines such as flunitrazepam are known to be extensively metabolized via N-dealkylation to active metabolites and via nitro reduction to the 7-amino compounds. The aim of the present work was to tentatively identify phase I and II metabolites of the latest members of this class appearing on the NPS market, clonazolam, meclonazepam, and nifoxipam, in human urine samples. Nano-liquid chromatography-high-resolution mass spectrometry was used to provide data about their detectability in urine. Data revealed that clonazolam and meclonazepam were extensively metabolized and mainly excreted as their amino and acetamino metabolites. Nifoxipam was also extensively metabolized, but instead mainly excreted as the acetamino metabolite and a glucuronic acid conjugate of the parent. Based on analysis of human urine samples collected in cases of acute intoxication within the Swedish STRIDA project, and samples submitted for routine drug testing, the most abundant metabolites and good targets for urine drug testing were 7-aminoclonazolam for clonazolam, 7-acetaminomeclonazepam for meclonazepam, and 7-acetaminonifoxipam for nifoxipam.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. UNODC. World Drug Report United Nations publication Sales No. E.14.XI.7. 2014.

  2. EMCDDA. New psychoactive substances in Europe. EMCDDA Annual Report, 2015.

  3. UNODC. World Drug Report United Nations publication Sales No. E.15.XI.6, 2015.

  4. Schifano F, Orsolini L, Duccio Papanti G, Corkery JM. Novel psychoactive substances of interest for psychiatry. World Psychiatry. 2015;14(1):15–26.

    Article  Google Scholar 

  5. Helander A, Beck O, Backberg M. Intoxications by the dissociative new psychoactive substances diphenidine and methoxphenidine. Clin Toxicol. 2015;53(5):446–53.

    Article  CAS  Google Scholar 

  6. Helander A, Backberg M, Beck O. MT-45, a new psychoactive substance associated with hearing loss and unconsciousness. Clin Toxicol. 2014;52(8):901–4.

    Article  CAS  Google Scholar 

  7. Backberg M, Lindeman E, Beck O, Helander A. Characteristics of analytically confirmed 3-MMC-related intoxications from the Swedish STRIDA project. Clin Toxicol. 2015;53(1):46–53.

    Article  Google Scholar 

  8. Backberg M, Beck O, Jonsson KH, Helander A. Opioid intoxications involving butyrfentanyl, 4-fluorobutyrfentanyl, and fentanyl from the Swedish STRIDA project. Clin Toxicol. 2015;53(7):609–17.

    Article  Google Scholar 

  9. Backberg M, Beck O, Hulten P, Rosengren-Holmberg J, Helander A. Intoxications of the new psychoactive substance 5-(2-aminopropyl)indole (5-IT): a case series from the Swedish STRIDA project. Clin Toxicol. 2014;52(6):618–24.

    Article  CAS  Google Scholar 

  10. Moosmann B, King LA, Auwarter V. Designer benzodiazepines: a new challenge. World Psychiatry. 2015;14(2):248.

    Article  Google Scholar 

  11. Moosmann B, Hutter M, Huppertz LM, Ferlaino S, Redlingshofer L, Auwarter V. Characterization of the designer benzodiazepine pyrazolam and its detectability in human serum and urine. Forensic Toxicol. 2013;31(2):263–71.

    Article  CAS  Google Scholar 

  12. Moosmann B, Huppertz LM, Hutter M, Buchwald A, Ferlaino S, Auwarter V. Detection and identification of the designer benzodiazepine flubromazepam and preliminary data on its metabolism and pharmacokinetics. J Mass Spectrom. 2013;48(11):1150–9.

    Article  CAS  Google Scholar 

  13. Moosmann B, Bisel P, Auwarter V. Characterization of the designer benzodiazepine diclazepam and preliminary data on its metabolism and pharmacokinetics. Drug Test Anal. 2014;6(7–8):757–63.

    Article  CAS  Google Scholar 

  14. Helander A, Beck O, Hagerkvist R, Hulten P. Identification of novel psychoactive drug use in Sweden based on laboratory analysis—initial experiences from the STRIDA project. Scand J Clin Lab Invest. 2013;73(5):400–6.

    Article  CAS  Google Scholar 

  15. Helander A, Backberg M, Hulten P, Al-Saffar Y, Beck O. Detection of new psychoactive substance use among emergency room patients: results from the Swedish STRIDA project. Forensic Sci Int. 2014;243:23–9.

    Article  CAS  Google Scholar 

  16. Danneberg P, Weber KH. Chemical structure and biological activity of the diazepines. Br J Clin Pharmacol. 1983;16 Suppl 2:231S–44S.

    Article  Google Scholar 

  17. Menezes CM, Rivera G, Alves MA, do Amaral DN, Thibaut JP, Noel F, et al. Synthesis, biological evaluation, and structure-activity relationship of clonazepam, meclonazepam, and 1,4-benzodiazepine compounds with schistosomicidal activity. Chem Biol Drug Des. 2012;79(6):943–9.

    Article  CAS  Google Scholar 

  18. Mahajan A, Kumar V, Mansour NR, Bickle Q, Chibale K. Meclonazepam analogues as potential new antihelmintic agents. Bioorg Med Chem Lett. 2008;18(7):2333–6.

    Article  CAS  Google Scholar 

  19. elSohly MA, Feng S, Salamone SJ, Wu R. A sensitive GC-MS procedure for the analysis of flunitrazepam and its metabolites in urine. J Anal Toxicol. 1997;21(5):335–40.

    Article  CAS  Google Scholar 

  20. Hoiseth G, Middelkoop G, Morland J, Gjerde H. Has previous abuse of flunitrazepam been replaced by clonazepam? Eur Addict Res. 2015;21(4):217–21.

    Article  Google Scholar 

  21. Druid H, Holmgren P, Ahlner J. Flunitrazepam: an evaluation of use, abuse and toxicity. Forensic Sci Int. 2001;122(2–3):136–41.

    Article  CAS  Google Scholar 

  22. Jones JD, Mogali S, Comer SD. Polydrug abuse: a review of opioid and benzodiazepine combination use. Drug Alcohol Depend. 2012;125(1–2):8–18.

    Article  CAS  Google Scholar 

  23. LinWu SW, Wu CA, Peng FC, Wang AH. Structure-based development of bacterial nitroreductase against nitrobenzodiazepine-induced hypnosis. Biochem Pharmacol. 2012;83(12):1690–9.

    Article  CAS  Google Scholar 

  24. Greenblatt DJ, Shader RI, Divoll M, Harmatz JS. Benzodiazepines: a summary of pharmacokinetic properties. Br J Clin Pharmacol. 1981;11 Suppl 1:11S–6S.

    Article  CAS  Google Scholar 

  25. Breimer DD. Pharmacokinetics and metabolism of various benzodiazepines used as hypnotics. Br J Clin Pharmacol. 1979;8(1):7S–13S.

    Article  CAS  Google Scholar 

  26. Huppertz LM, Bisel P, Westphal F, Franz F, Auwarter V, Moosmann B. Characterization of the four designer benzodiazepines clonazolam, deschloroetizolam, flubromazolam, and meclonazepam, and identification of their in vitro metabolites. Forensic Toxicol. 2015;33(2):388–95.

    Article  CAS  Google Scholar 

  27. Spaggiari D, Geiser L, Rudaz S. Coupling ultra-high-pressure liquid chromatography with mass spectrometry for in-vitro drug-metabolism studies. Trac Trend Anal Chem. 2014;63:129–39.

    Article  CAS  Google Scholar 

  28. Rainville PD, Smith NW, Wilson ID, Nicholson JK, Plumb RS. Addressing the challenge of limited sample volumes in in vitro studies with capillary-scale microfluidic LC-MS/MS. Bioanalysis. 2011;3(8):873–82.

    Article  CAS  Google Scholar 

  29. Valaskovic GA, Utley L, Lee MS, Wu JT. Ultra-low flow nanospray for the normalization of conventional liquid chromatography/mass spectrometry through equimolar response: standard-free quantitative estimation of metabolite levels in drug discovery. Rapid Commun Mass Spectrom. 2006;20(7):1087–96.

    Article  CAS  Google Scholar 

  30. Wickremsinhe ER, Singh G, Ackermann BL, Gillespie TA, Chaudhary AK. A review of nanoelectrospray ionization applications for drug metabolism and pharmacokinetics. Curr Drug Metab. 2006;7(8):913–28.

    Article  CAS  Google Scholar 

  31. Schadt S, Chen LZ, Bischoff D. Evaluation of relative LC/MS response of metabolites to parent drug in LC/nanospray ionization mass spectrometry: potential implications in MIST assessment. J Mass Spectrom. 2011;46(12):1281–6.

    Article  Google Scholar 

  32. Forsman M, Nystrom I, Roman M, Berglund L, Ahlner J, Kronstrand R. Urinary detection times and excretion patterns of flunitrazepam and its metabolites after a single oral dose. J Anal Toxicol. 2009;33(8):491–501.

    Article  CAS  Google Scholar 

  33. Negrusz A, Moore CM, Stockham TL, Poiser KR, Kern JL, Palaparthy R, et al. Elimination of 7-aminoflunitrazepam and flunitrazepam in urine after a single dose of Rohypnol. J Forensic Sci. 2000;45(5):1031–40.

    Article  CAS  Google Scholar 

  34. Coassolo P, Aubert C, Cano JP. Plasma determination of 3-methylclonazepam by capillary gas chromatography. J Chromatogr. 1985;338(2):347–55.

    Article  CAS  Google Scholar 

  35. Niessen WM. Fragmentation of toxicologically relevant drugs in positive-ion liquid chromatography-tandem mass spectrometry. Mass Spectrom Rev. 2011;30(4):626–63.

    Article  CAS  Google Scholar 

  36. Smyth WF, McClean S, Ramachandran VN. A study of the electrospray ionisation of pharmacologically significant 1,4-benzodiazepines and their subsequent fragmentation using an ion-trap mass spectrometer. Rapid Commun Mass Spectrom. 2000;14(21):2061–9.

    Article  CAS  Google Scholar 

  37. Smyth WF, Joyce C, Ramachandran VN, O’ Kane E, Coulter D. Characterisation of selected hypnotic drugs and their metabolites using electrospray ionisation with ion trap mass spectrometry and with quadrupole time-of-flight mass spectrometry and their determination by liquid chromatography-electrospray ionisation-ion trap mass spectrometry. Anal Chim Acta. 2004;506(2):203–14.

    Article  CAS  Google Scholar 

  38. Bourcier K, Hyland R, Kempshall S, Jones R, Maximilien J, Irvine N, et al. Investigation into UDP-glucuronosyltransferase (UGT) enzyme kinetics of imidazole- and triazole-containing antifungal drugs in human liver microsomes and recombinant UGT enzymes. Drug Metab Dispos. 2010;38(6):923–9.

    Article  CAS  Google Scholar 

  39. Court MH, Hao Q, Krishnaswamy S, Bekaii-Saab T, Al-Rohaimi A, von Moltke LL, et al. UDP-glucuronosyltransferase (UGT) 2B15 pharmacogenetics: UGT2B15 D85Y genotype and gender are major determinants of oxazepam glucuronidation by human liver. J Pharmacol Exp Ther. 2004;310(2):656–65.

    Article  CAS  Google Scholar 

  40. Meyer MR, Robert A, Maurer HH. Toxicokinetics of novel psychoactive substances: characterization of N-acetyltransferase (NAT) isoenzymes involved in the phase II metabolism of 2C designer drugs. Toxicol Lett. 2014;227(2):124–8.

    Article  CAS  Google Scholar 

  41. Schwaninger AE, Meyer MR, Zapp J, Maurer HH. The role of human UDP-glucuronyltransferases on the formation of the methylenedioxymethamphetamine (ecstasy) phase II metabolites R- and S-3-methoxymethamphetamine 4-O-glucuronides. Drug Metab Dispos. 2009;37(11):2212–20.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The STRIDA project was supported by grants from the Public Health Agency of Sweden.

Author information

Author notes

  1. Markus R. Meyer

    Present address: Department of Pharmacology and Pharmacoepidemiology, Heidelberg University Hospital, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany

Authors and Affiliations

  1. Karolinska Institutet, Department of Laboratory Medicine, 141 86, Stockholm, Sweden

    Markus R. Meyer, Madeleine Pettersson Bergstrand, Anders Helander & Olof Beck

  2. Department of Clinical Pharmacology, Karolinska University Laboratory, Huddinge, 141 86, Stockholm, Sweden

    Anders Helander & Olof Beck

Authors
  1. Markus R. Meyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Madeleine Pettersson Bergstrand
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anders Helander
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Olof Beck
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Markus R. Meyer.

Ethics declarations

The study was conducted in accordance with the Helsinki Declaration. The use of de-identified leftover volumes from the routine samples pool (No. 00-230) and the STRIDA project (No. 2013/116–31/2) were approved by the regional ethical review board.

Conflict of interest

The authors declare that they have no conflicts of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 112 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Meyer, M.R., Bergstrand, M.P., Helander, A. et al. Identification of main human urinary metabolites of the designer nitrobenzodiazepines clonazolam, meclonazepam, and nifoxipam by nano-liquid chromatography-high-resolution mass spectrometry for drug testing purposes. Anal Bioanal Chem 408, 3571–3591 (2016). https://doi.org/10.1007/s00216-016-9439-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9439-6

Keywords

Search

Navigation