Environmental Chemistry Letters

, Volume 13, Issue 2, pp 203–210 | Cite as

Dispersive liquid–liquid microextraction and HPLC to analyse fluoxetine and metoprolol enantiomers in wastewaters

  • Ana R. Ribeiro
  • Virgínia M. F. Gonçalves
  • Alexandra S. Maia
  • Cláudia Ribeiro
  • Paula M. L. Castro
  • Maria E. Tiritan
Original Paper


Sample extraction is a major step in environmental analyses due both to the high complexity of matrices and to the low concentration of the target analytes. Sample extraction is usually expensive, laborious, time-consuming and requires a high amount of organic solvents. Actually, there is a lack of miniaturized methodologies for sample extraction and chiral analyses. Here, we developed a dispersive liquid–liquid microextraction (DLLME) to extract the pharmaceuticals fluoxetine and metoprolol, as models of basic chiral compounds, from wastewater samples. Compounds were then analysed by enantioselective high-performance liquid chromatography. We monitored the influence of sample pH, extracting and dispersive solvent and respective volumes, salt addition, extracting and vortexing time. The DLLME method was validated within the range of 1–10 µg L−1 for fluoxetine enantiomers and 0.5–10 µg L−1 for metoprolol enantiomers. Accuracy ranged from 90.6 to 106 % and recovery rates from 54.5 to 81.5 %. Relative standard deviation values lower than 7.84 and 9.00 % were obtained for intra- and inter-batch precision, respectively.


Dispersive liquid–liquid microextraction Sample preparation HPLC-FD Chiral pharmaceuticals Chirobiotic V Wastewaters 



The work has been supported by Fundação para a Ciência e Tecnologia—FCT (PhD. grant attributed to ARR and ASM respectively, SFRH/BD/64999/2009 and SFRH/BD/86939/2012), from QREN-POPH, European Social Fund and MCTES. Authors also wish to acknowledge the support from national funds from FCT through projects FLUOROPHARMA, (PTDC/EBB-EBI/111699/2009), PEst-OE/EQB/LA0016/2013 and CEQUIMED-PEst-OE/SAU/UI4040/2014, as well as the support from CESPU through project PHARMADRUGS-CESPU-2014.


  1. Al Aukidy M, Verlicchi P, Voulvoulis N (2014) A framework for the assessment of the environmental risk posed by pharmaceuticals originating from hospital effluents. Sci Total Environ 493:54–64CrossRefGoogle Scholar
  2. Barclay VKH, Tyrefors NL, Johansson IM, Pettersson CE (2012a) Chiral analysis of metoprolol and two of its metabolites, α-hydroxymetoprolol and deaminated metoprolol, in wastewater using liquid chromatography–tandem mass spectrometry. J Chromatogr A 1269:208–217CrossRefGoogle Scholar
  3. Barclay VKH, Tyrefors NL, Johansson IM, Pettersson CE (2012b) Trace analysis of fluoxetine and its metabolite norfluoxetine. Part II: enantioselective quantification and studies of matrix effects in raw and treated wastewater by solid phase extraction and liquid chromatography–tandem mass spectrometry. J Chromatogr A 1227:105–114CrossRefGoogle Scholar
  4. Brooks BW, Turner PK, Stanley JK, Weston JJ, Glidewell EA, Foran CM, Slattery M, La Point TW, Huggett DB (2003) Waterborne and sediment toxicity of fluoxetine to select organisms. Chemosphere 52(1):135–142CrossRefGoogle Scholar
  5. Buser H-R, Poiger T, Muller MD (1999) Occurrence and environmental behavior of the chiral pharmaceutical drug ibuprofen in surface waters and in wastewater. Environ Sci Technol 33(15):2529–2535CrossRefGoogle Scholar
  6. Daughton CG (2014) Eco-directed sustainable prescribing: feasibility for reducing water contamination by drugs. Sci Total Environ 493:392–404CrossRefGoogle Scholar
  7. De Andrés F, Castañeda G, Ríos Á (2009) Use of toxicity assays for enantiomeric discrimination of pharmaceutical substances. Chirality 21(8):751–759CrossRefGoogle Scholar
  8. de la Guardia M, Garrigues S (2014) The social responsibility of environmental analysis. Trends Environ Anal Chem 3–4:7–13CrossRefGoogle Scholar
  9. Dong Z, Senn DB, Moran RE, Shine JP (2013) Prioritizing environmental risk of prescription pharmaceuticals. Regul Toxicol Pharmacol 65(1):60–67CrossRefGoogle Scholar
  10. FDA (2001). Bioanalytical method validation: guidance for industry, U.S. Food and Drug Administration: 1–22.
  11. Flaherty CM, Dodson SI (2005) Effects of pharmaceuticals on Daphnia survival, growth, and reproduction. Chemosphere 61(2):200–207CrossRefGoogle Scholar
  12. Fono LJ, Sedlak DL (2005) Use of the chiral pharmaceutical propranolol to identify sewage discharges into surface waters. Environ Sci Technol 39(23):9244–9252CrossRefGoogle Scholar
  13. Foran CM, Weston J, Slattery M, Brooks BW, Huggett DB (2004) Reproductive assessment of Japanese Medaka (Oryzias latipes) following a four-week fluoxetine (SSRI) exposure. Arch Environ Contam Toxicol 46(4):511–517CrossRefGoogle Scholar
  14. Gonzalez-Rey M, Bebianno MJ (2013) Does selective serotonin reuptake inhibitor (SSRI) fluoxetine affects mussel Mytilus galloprovincialis? Environ Pollut 173:200–209CrossRefGoogle Scholar
  15. Henry TB, Black MC (2008) Acute and chronic toxicity of fluoxetine (selective serotonin reuptake inhibitor) in western mosquitofish. Arch Environ Contam Toxicol 54(2):325–330CrossRefGoogle Scholar
  16. Herrera-Herrera AV, Hernández-Borges J, Borges-Miquel TM, Rodríguez-Delgado MÁ (2013) Dispersive liquid–liquid microextraction combined with ultra-high performance liquid chromatography for the simultaneous determination of 25 sulfonamide and quinolone antibiotics in water samples. J Pharm Biomed Anal 75:130–137CrossRefGoogle Scholar
  17. Hughes SR, Kay P, Brown LE (2013) Global synthesis and critical evaluation of pharmaceutical data sets collected from river systems. Environ Sci Technol 47(2):661–677CrossRefGoogle Scholar
  18. ICH (1996) Validation of analytical procedures: text and methodology Q2(R1). International Conference on Harmonization (March 3):1–13Google Scholar
  19. Lao W, Gan J (2012) Enantioselective degradation of warfarin in soils. Chirality 24(1):54–59CrossRefGoogle Scholar
  20. Lima DLD, Silva CP, Otero M, Esteves VI (2013) Low cost methodology for estrogens monitoring in water samples using dispersive liquid–liquid microextraction and HPLC with fluorescence detection. Talanta 115:980–985CrossRefGoogle Scholar
  21. Lima DLD, Silva CP, Schneider RJ, Otero M, Esteves VI (2014) Application of dispersive liquid–liquid microextraction for estrogens' quantification by enzyme-linked immunosorbent assay. Talanta 125:102–106CrossRefGoogle Scholar
  22. López-Serna R, Kasprzyk-Hordern B, Petrović M, Barceló D (2013) Multi-residue enantiomeric analysis of pharmaceuticals and their active metabolites in the Guadalquivir River basin (South Spain) by chiral liquid chromatography coupled with tandem mass spectrometry. Anal Bioanal Chem 405(18):5859–5873CrossRefGoogle Scholar
  23. MacLeod SL, Sudhir P, Wong CS (2007) Stereoisomer analysis of wastewater-derived b-blockers, selective serotonin re-uptake inhibitors, and salbutamol by high-performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1170(1–2):23–33CrossRefGoogle Scholar
  24. Martín J, Buchberger W, Alonso E, Himmelsbach M, Aparicio I (2011) Comparison of different extraction methods for the determination of statin drugs in wastewater and river water by HPLC/Q-TOF-MS. Talanta 85(1):607–615CrossRefGoogle Scholar
  25. Maszkowska J, Stolte S, Kumirska J, Lukaszewicz P, Mioduszewska K, Puckowski A, Caban M, Wagil M, Stepnowski P, Bialk-Bielinska A (2014) Beta-blockers in the environment: part II. Ecotoxicity study. Sci Total Environ 493:1122–1126CrossRefGoogle Scholar
  26. Morando MB, Medeiros LR, McDonald MD (2009) Fluoxetine treatment affects nitrogen waste excretion and osmoregulation in a marine teleost fish. Aquat Toxicol 95(2):164–171CrossRefGoogle Scholar
  27. Morante-Zarcero S, Sierra I (2012) Simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural waters by HPLC on new polysaccharide-based stationary phase using a highly selective molecularly imprinted polymer extraction. Chirality 24(10):860–866CrossRefGoogle Scholar
  28. Paterson G, Metcalfe CD (2008) Uptake and depuration of the anti-depressant fluoxetine by the Japanese medaka (Oryzias latipes). Chemosphere 74(1):125–130CrossRefGoogle Scholar
  29. Rezaee M, Assadi Y, Milani Hosseini M-R, Aghaee E, Ahmadi F, Berijani S (2006) Determination of organic compounds in water using dispersive liquid–liquid microextraction. J Chromatogr A 1116(1–2):1–9CrossRefGoogle Scholar
  30. Ribeiro AR, Castro PML, Tiritan ME (2012) Chiral pharmaceuticals in the environment. Environ Chem Lett 10(3):239–253CrossRefGoogle Scholar
  31. Ribeiro AR, Afonso CM, Castro PML, Tiritan ME (2013) Enantioselective HPLC analysis and biodegradation of atenolol, metoprolol and fluoxetine. Environ Chem Lett 11(1):83–90CrossRefGoogle Scholar
  32. Ribeiro AR, Maia AS, Cass QB, Tiritan ME (2014a) Enantioseparation of chiral pharmaceuticals in biomedical and environmental analyses by liquid chromatography: an overview. J Chromatogr B 968:8–21CrossRefGoogle Scholar
  33. Ribeiro AR, Santos LHMLM, Maia AS, Delerue-Matos C, Castro PML, Tiritan ME (2014b) Enantiomeric fraction evaluation of pharmaceuticals in environmental matrices by liquid chromatography-tandem mass spectrometry. J Chromatogr A 1363:226–235CrossRefGoogle Scholar
  34. Ribeiro C, Ribeiro AR, Maia AS, Gonçalves VMF, Tiritan ME (2014c) New trends in sample preparation techniques for environmental analysis. Crit Rev Anal Chem 44(2):142–185CrossRefGoogle Scholar
  35. Sánchez-Argüello P, Fernández C, Tarazona JV (2009) Assessing the effects of fluoxetine on Physa acuta (Gastropoda, Pulmonata) and Chironomus riparius (Insecta, Diptera) using a two-species water-sediment test. Sci Total Environ 407(6):1937–1946CrossRefGoogle Scholar
  36. Santos LHMLM, Gros M, Rodriguez-Mozaz S, Delerue-Matos C, Pena A, Barceló D, Montenegro MCBSM (2013) Contribution of hospital effluents to the load of pharmaceuticals in urban wastewaters: identification of ecologically relevant pharmaceuticals. Sci Total Environ 461–462:302–316CrossRefGoogle Scholar
  37. Schultz MM, Painter MM, Bartell SE, Logue A, Furlong ET, Werner SL, Schoenfuss HL (2011) Selective uptake and biological consequences of environmentally relevant antidepressant pharmaceutical exposures on male fathead minnows. Aquat Toxicol 104(1–2):38–47CrossRefGoogle Scholar
  38. Silvestre CIC, Santos JLM, Lima JLFC, Zagatto EAG (2009) Liquid–liquid extraction in flow analysis: a critical review. Anal Chim Acta 652(1–2):54–65CrossRefGoogle Scholar
  39. Stanley JK, Brooks BW (2009) Perspectives on ecological risk assessment of chiral compounds. Integr Environ Assess Manag 5(3):364–373CrossRefGoogle Scholar
  40. Vázquez MdMP, Vázquez PP, Galera MM, Sánchez LM (2012) Simple, rapid, and sensitive determination of beta-blockers in environmental water using dispersive liquid–liquid microextraction followed by liquid chromatography with fluorescence detection. J Sep Sci 35(17):2184–2192CrossRefGoogle Scholar
  41. Vulliet E, Cren-Olivé C, Grenier-Loustalot M-F (2011) Occurrence of pharmaceuticals and hormones in drinking water treated from surface waters. Environ Chem Lett 9(1):103–114CrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Ana R. Ribeiro
    • 1
    • 2
    • 3
    • 4
  • Virgínia M. F. Gonçalves
    • 1
  • Alexandra S. Maia
    • 1
    • 2
  • Cláudia Ribeiro
    • 1
  • Paula M. L. Castro
    • 2
  • Maria E. Tiritan
    • 1
    • 3
    • 5
  1. 1.CESPUInstituto de Investigação e Formação Avançada em Ciências e Tecnologias da SaúdeGandraPortugal
  2. 2.CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de BiotecnologiaUniversidade Católica Portuguesa/PortoPortoPortugal
  3. 3.Centro de Química Medicinal da Universidade do Porto (CEQUIMED-UP), Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de FarmáciaUniversidade do PortoPortoPortugal
  4. 4.LCM – Laboratory of Catalysis and Materials – Associate Laboratory LSRE-LCMFaculdade de Engenharia, Universidade do PortoPortoPortugal
  5. 5.Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR/CIMAR)Universidade do PortoPortoPortugal

Personalised recommendations