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Highly sensitive determination of 68 psychoactive pharmaceuticals, illicit drugs, and related human metabolites in wastewater by liquid chromatography–tandem mass spectrometry


The present work describes the development and validation of a highly sensitive analytical method for the simultaneous determination of 68 compounds, including illicit drugs (opiates, opioids, cocaine compounds, amphetamines, and hallucinogens), psychiatric drugs (benzodiazepines, barbiturates, anesthetics, antiepileptics, antipsychotics, antidepressants, and sympathomimetics), and selected human metabolites in influent and effluent wastewater (IWW and EWW) by liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS). The method involves a pre-concentration and cleanup step, carried out by solid-phase extraction (SPE) using the adsorbent Strata-XC, followed by the instrumental analysis performed by LC–MS/MS, using a Kinetex pentafluorophenyl (PFP) reversed-phase fused-core column and electrospray ionization (ESI) in both positive and negative modes. A systematic optimization of mobile phases was performed to cope with the wide range of physicochemical properties of the analytes. The PFP column was also compared with two reversed-phase columns: fused-core C18 and XB-C18 (with a cross-butyl C18 ligand). SPE optimization and critical aspects associated with the trace level determination of the target compounds (e.g., matrix effects) have been also considered and discussed. Fragmentation patterns for all the classes were proposed. The validated method provides absolute recoveries between 75 and 120 % for most compounds in IWW and EWW. Low method limits of detection were achieved (between 0.04 and 10.0 ng/L for 87 % of the compounds), allowing a reliable and accurate quantification of the analytes at trace level. The method was successfully applied to the analysis of these compounds in five wastewater treatment plants in Santorini, a touristic island of the Aegean Sea, Greece. Thirty-two out of 68 compounds were detected in all IWW samples in the range between 0.6 ng/L (for nordiazepam) and 6,822 ng/L (for carbamazepine) and 22 out of 68 in all EWW samples, with values between 0.4 ng/L (for 9-OH risperidone) and 2,200 ng/L (for carbamazepine). The novel methodology described herein maximizes the information on the environmental analysis of these substances and also provides a first profile of 68 drugs in a Greek touristic area.

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  1. 1.

    Aldrich MR (2012) Antique cannabis book, medical cannabis: a short graphical history, [Accessed on 12 Oct 2013]

  2. 2.

    ΕMCDDA (European Monitoring Centre for Drugs and Drug Addiction) (2012) Annual report: the state of the drug problem in Europe. [Accessed on 12 Jan 2014]

  3. 3.

    Kyriopoulos J, Tsiantou V (2010) Arch Hellen Med 27:834–840

    Google Scholar 

  4. 4.

    Stuckler D, Basu S, Suhrcke M, Coutts A, McKee M (2009) Lancet 374:315–323

    Article  Google Scholar 

  5. 5.

    Daughton CG (2011) In: Castiglioni S, Zuccato E, Fanelli R (eds) Illicit drugs in the environment: occurrence, analysis and fate using mass spectrometry. Canada, Wiley

    Google Scholar 

  6. 6.

    Van Nuijs ALN, Mougel JF, Taracomnicu I, Bervoets L, Blust R, Jorens PG, Neels H, Covaci A (2011) J Environ Monitor 13:1008–1016

    Article  Google Scholar 

  7. 7.

    Pal R, Megharaj M, Kirkbride KP, Naidu R (2013) Sci Total Environ 463–464:1079–1092

    Article  Google Scholar 

  8. 8.

    Jones-Lepp TL, Alvarez DA, Petty JD, Huckins JN (2004) Arch Environ Contam Toxicol 47:427–439

    CAS  Article  Google Scholar 

  9. 9.

    Boles TH, Wells MJM (2010) J Chromatogr A 1217:2561–2568

    CAS  Article  Google Scholar 

  10. 10.

    Zuccato E, Castiglioni S (2009) Phil Trans R Soc A 367:3965–3978

    CAS  Article  Google Scholar 

  11. 11.

    Zuccato E, Chiabrando C, Castiglioni S, Calamari D, Bagnati R, Schiarea S, Fanelli R (2005) Environ Health 4:14

    Article  Google Scholar 

  12. 12.

    Zuccato E, Castiglioni S, Bagnati R, Chiabrando C, Grassi P, Fanelli R (2008) Water Res 42:961–968

    CAS  Article  Google Scholar 

  13. 13.

    Daughton CG, Ruhoy IS (2009) Environ Toxicol Chem 28:2495–2521

    CAS  Article  Google Scholar 

  14. 14.

    Moore TJ, Glenmullen J, Furberg CD (2010) PLoS One 5:e15337

    CAS  Article  Google Scholar 

  15. 15.

    Binelli A, Pedriali A, Riva C, Parolini M (2012) Chemosphere 86:906–911

    CAS  Article  Google Scholar 

  16. 16.

    Hummel D, Löffler D, Fink G, Ternes TA (2006) Environ Sci Technol 40:7321–7328

    CAS  Article  Google Scholar 

  17. 17.

    Postigo C, López de Alda MJ, Barceló D (2008) Anal Chem 80:3123–3314

    CAS  Article  Google Scholar 

  18. 18.

    Postigo C, López de Alda MJ, Barceló D (2010) Environ Int 36:75–84

  19. 19.

    Postigo C, López de Alda MJ, Barceló D (2011) Environ Int 37:49–55

  20. 20.

    Huerta-Fontela M, Galceran MT, Ventura F (2008) Environ Technol 42:6809–6816

    CAS  Article  Google Scholar 

  21. 21.

    Huerta-Fontela M, Galceran MT, Ventura F (2007) Anal Chem 79:3821–3829

    CAS  Article  Google Scholar 

  22. 22.

    Gonzales Alonso S, Catala M, Maroto RR, Rodrigez Gill JL, De Miguel AG, Valcarcel Y (2010) Environ Int 36:195–201

    Article  Google Scholar 

  23. 23.

    Vazquez-Roing P, Andreu V, Blasco C, Pico Y (2010) Anal Bioanal Chem 397:2851–2864

    Article  Google Scholar 

  24. 24.

    González-Mariño I, Quintana JB, Rodriguez I, Cela R (2010) J Chromatogr A 1217:1748–1760

    Article  Google Scholar 

  25. 25.

    Bijlsma L, Sancho JV, Pitarch E, Ibáñez M, Hernández F (2009) J Chromatogr A 1216:3078–3089

    CAS  Article  Google Scholar 

  26. 26.

    Hernández F, Bijlsma L, Sancho VJ, Diaz R, Ibáñez M (2011) Anal Chim Acta 684:96–106

    Article  Google Scholar 

  27. 27.

    Boleda MR, Galceran MT, Ventura F (2007) J Chromatogr A 1175:38–48

    CAS  Article  Google Scholar 

  28. 28.

    Boleda MR, Galceran MT, Ventura F (2009) Water Res 43:1126–1136

    CAS  Article  Google Scholar 

  29. 29.

    Bueno MJM, Uclés S, Hernando MD, Fernández-Alba AR (2011) Talanta 85:157–166

    Article  Google Scholar 

  30. 30.

    Bisceglia KJ, Roberts AL, Schantz MM, Lippa KL (2010) Anal Bioanal Chem 398:2701–2712

    CAS  Article  Google Scholar 

  31. 31.

    Chiaia AC, Banta-Green C, Field J (2008) Environ Sci Technol 42:8841–8848

    CAS  Article  Google Scholar 

  32. 32.

    Metcalfe C, Tindale K, Li H, Rodayan A, Yargeau V (2010) Environ Pollut 158:3179–3185

    CAS  Article  Google Scholar 

  33. 33.

    Karolak S, Nefau T, Bailly E, Solgadi A, Levi Y (2010) Forensic Sci Int 200:153–160

    CAS  Article  Google Scholar 

  34. 34.

    Bones J, Kevin VT, Brett P (2007) J Environ Monit 9:701–707

    CAS  Article  Google Scholar 

  35. 35.

    Van Nuijs ALN, Pecceu B, Theunis L, Dubois N, Charlier C, Jorens PG, Bervoets L, Blust R, Meulemans H, Neels H, Covaci A (2009) Addiction 104:734–742

    Article  Google Scholar 

  36. 36.

    Van Nuijs ALN, Tarcomnicu I, Bervoets L, Blust R, Jorens PG, Neels H, Covaci A (2009) Anal Bioanal Chem 395:819–828

    Article  Google Scholar 

  37. 37.

    Kasprzyk-Hordern B, Dinsdale RM, Guwy AJ (2008) Water Res 42:3498–3518

    CAS  Article  Google Scholar 

  38. 38.

    Bakera DR, Kasprzyk-Hordern B (2011) J Chromatogr A 1218:1620–1631

    Article  Google Scholar 

  39. 39.

    Kasprzyk-Hordern B, Dinsdale RM, Guwy AJ (2009) Environ Pol 157:1773–1777

    CAS  Article  Google Scholar 

  40. 40.

    Bijlsma L, Emke E, Hernández F, De Voogt P (2012) Chemosphere 89:1399–1406

    CAS  Article  Google Scholar 

  41. 41.

    Zuccato E, Castiglioni S, Tettamanti M, Olandese R, Bagnati R, Melis M, Fanelli R (2011) Drug Alcohol Depend 118:464–469

    Article  Google Scholar 

  42. 42.

    Berset JD, Brenneisen R, Mathieu C (2010) Chemosphere 81:859–866

    CAS  Article  Google Scholar 

  43. 43.

    Irvine RJ, Kostakis C, Felgate PD, Jaehne EJ, Chen C, White JM (2011) Forensic Sci Int 210:69–73

    CAS  Article  Google Scholar 

  44. 44.

    Terzic S, Senta I, Ahel M (2010) Environ Pollut 158:2686–2693

    CAS  Article  Google Scholar 

  45. 45.

    Chemicalize, [Accessed on 12 Jan 2014]

  46. 46.

    Peel MC, Finlayson BL, McMahon TA (2007) Earth Syst Sci Discuss 4:439–473

    Article  Google Scholar 

  47. 47.

    van Nuijs ALN, Abdellati K, Bervoets L, Blust R, Jorens PG, Neels H, Covaci A (2012) J Hazard Mater 239–240:19–23

    Article  Google Scholar 

  48. 48.

    Baker DR, Kasprzyk-Hordern B (2011) J Chromatogr A 1218:8036–8059

    CAS  Article  Google Scholar 

  49. 49.

    Chemspider, [Accessed on 12 Jan 2013]

  50. 50.

    Huq S, Dixon A, Kelly K, Kallury KMR (2005) J Chromatogr A 1073:355–361

    CAS  Article  Google Scholar 

  51. 51.

    González-Mariño I, Quintana JB, Rodríguez I, Gonzáez-Díez M, Cela R (2012) Anal Chem 84:1708–1717

    Article  Google Scholar 

  52. 52.

    Kot-Wasik A, Debska J, Namiesnik J (2007) Trends in Anal Chem 26:557–568

    CAS  Article  Google Scholar 

  53. 53.

    Dams R, Huestis MA, Lambert WE, Murphy CM (2003) J Am Soc Mass Spectrom 14:1290–1294

    CAS  Article  Google Scholar 

  54. 54.

    Lajeunesse A, Gagnon C, Sauve S (2008) Anal Chem 80:5325–5333

    CAS  Article  Google Scholar 

  55. 55.

    Jelic A, Gros M, Ginebreda A, Cespedes-Sánchez R, Ventura F, Petrovic M, Barcelo D (2011) Water Res 45:1165–1176

    CAS  Article  Google Scholar 

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This project was implemented under the Operational Program “Education and Lifelong Learning” and funded by the European Union (European Social Fund) and National Resources–ARISTEIA 624.

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Correspondence to Νikolaos S. Τhomaidis.

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Borova, V.L., Maragou, N.C., Gago-Ferrero, P. et al. Highly sensitive determination of 68 psychoactive pharmaceuticals, illicit drugs, and related human metabolites in wastewater by liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 406, 4273–4285 (2014).

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  • Illicit drugs
  • Psychiatric drugs
  • Wastewater
  • LC–MS/MS
  • SPE