Skip to main content
SpringerLink
Log in

Ultrasound assisted extraction combined with dispersive liquid–liquid microextraction (US-DLLME)—a fast new approach to measure phthalate metabolites in nails

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

Abstract

A new, fast, and environmentally friendly method based on ultrasound assisted extraction combined with dispersive liquid–liquid microextraction (US-DLLME) was developed and optimized for assessing the levels of seven phthalate metabolites (including the mono(ethyl hexyl) phthalate (MEHP), mono(2-ethyl-5-hydroxyhexyl) phthalate (5-OH-MEHP), mono(2-ethyl-5-oxohexyl) phthalate (5-oxo-MEHP), mono-n-butyl phthalate (MnBP), mono-isobutyl phthalate (MiBP), monoethyl phthalate (MEP), and mono-benzyl phthalate (MBzP)) in human nails by UPLC-MS/MS. The optimization of the US-DLLME method was performed using a Taguchi combinatorial design (L9 array). Several parameters such as extraction solvent, solvent volume, extraction time, acid, acid concentration, and vortex time were studied. The optimal extraction conditions achieved were 180 μL of trichloroethylene (extraction solvent), 2 mL trifluoroacetic acid in methanol (2 M), 2 h extraction and 3 min vortex time. The optimized method had a good precision (6–17 %). The accuracy ranged from 79 to 108 % and the limit of method quantification (LOQm) was below 14 ng/g for all compounds. The developed US-DLLME method was applied to determine the target metabolites in 10 Belgian individuals. Levels of the analytes measured in nails ranged between <12 and 7982 ng/g. The MEHP, MBP isomers, and MEP were the major metabolites and detected in every sample. Miniaturization (low volumes of organic solvents used), low costs, speed, and simplicity are the main advantages of this US-DLLME based method.

Extraction and phase separation of the US-DLLME procedure

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

Similar content being viewed by others

References

  1. Alves A, Kucharska A, Erratico C, Xu F, Den Hond E, Koppen G, et al. Human biomonitoring of emerging pollutants through noninvasive matrices: state of the art and future potential. Anal Bioanal Chem. 2014;406:4063–88.

    Article  CAS  Google Scholar 

  2. Braun JM, Just AC, Williams PL, Smith KW, Calafat AM, Hauser R. Personal care product use and urinary phthalate metabolite and paraben concentrations during pregnancy among women from a fertility clinic. J Exp Sci Environ Epidemiol. 2013;24:459–66.

    Article  Google Scholar 

  3. Adibi JJ, Perera FP, Jedrychowski W, Camann DE, Barr D, Jacek R, et al. Prenatal exposures to phthalates among women in New York City and Krakow, Poland. Environ Health Perspect. 2003;111:1719–22.

    Article  CAS  Google Scholar 

  4. Barr DB, Silva MJ, Kato K, Reidy JA, Malek NA, Hurtz D, et al. Assessing human exposure to phthalates using monoesters and their oxidized metabolites as biomarkers. Environ Health Perspect. 2003;111:1148–51.

    Article  Google Scholar 

  5. Wittassek M, Koch HM, Angerer J, Brüning T. Assessing exposure to phthalates—the human biomonitoring approach. Mol Nutr Food Res. 2011;55:7–31.

    Article  CAS  Google Scholar 

  6. Romero-Franco M, Hernández-Ramírez RU, Calafat AM, Cebrián ME, Needham L, Teitelbaum S, et al. Personal care product use and urinary levels of phthalate metabolites in Mexican women. Environ Int. 2011;37:867–71.

    Article  CAS  Google Scholar 

  7. Koch HM, Lorber M, Christensen KLY, Pälmke C, Koslitz S, Brüning T. Identifying sources of phthalate exposure with human biomonitoring: results of a 48h fasting study with urine collection and personal activity patterns. Int J Hyg Environ Health. 2013;216:672–81.

    Article  CAS  Google Scholar 

  8. Hauser R, Calafat AM. Phthalates and human health. Occup Environ Med. 2005;62:806–18.

    Article  CAS  Google Scholar 

  9. Foster PMD, Lake BG, Thomas LV, Cook MW, Gangolli SD. Studies on the testicular effects and zinc excretion produced by various isomers of monobutyl-ortho-phthalate in the rat. Chem Biol Interact. 1981;34:233–8.

    Article  CAS  Google Scholar 

  10. Saravanabhavan G, Murray J. Human biological monitoring of diisononyl phthalate and diisodecyl phthalate: a review. J Environ Public Health. 2012;2012:810501.

    Article  Google Scholar 

  11. Ventrice P, Ventrice D, Russo E, De Sarro G. Phthalates: European regulation, chemistry, pharmacokinetic, and related toxicity. Environ Toxicol Pharmacol. 2013;36:88–96.

    Article  CAS  Google Scholar 

  12. Koch HM, Christensen KLY, Harth V, Lorber M, Brüning T. Di-n-butyl phthalate (DnBP) and diisobutyl phthalate (DiBP) metabolism in a human volunteer after single oral doses. Arch Toxicol. 2012;86:1829–39.

    Article  CAS  Google Scholar 

  13. Peck CC, Albro PW. Toxic potential of the plasticizer Di(2-ethylhexyl) phthalate in the context of its disposition and metabolism in primates and man. Environ Health Perspect. 1982;45:11–7.

    Article  CAS  Google Scholar 

  14. Wittassek M, Angerer J. Phthalates: metabolism and exposure. Int J Androl. 2008;31:131–8.

    Article  CAS  Google Scholar 

  15. Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, et al. Levels of seven urinary phthalate metabolites in a human reference population. Environ Health Perspect. 2000;108:979–82.

    Article  CAS  Google Scholar 

  16. Bornehag C-G, Lundgren B, Weschler CJ, Singsgaard T, Hagerhed-Engman L, Sundell J. Phthalates in indoor dust and their association with building characteristics. Environ Health Perspect. 2005;113:1399–404.

    Article  CAS  Google Scholar 

  17. Abb M, Heinrich T, Sorkau E, Lorenz W. Phthalates in house dust. Environ Int. 2009;35:965–70.

    Article  CAS  Google Scholar 

  18. Directive 2005/90/EC relating to restrictions on the marketing and use of certain dangerous substances and preparations (substances classified as carcinogenic, mutagenic, or toxic to reproduction—c/m/r). L33/28

  19. Directive 2003/36/EC relating to restrictions on the marketing and use of certain dangerous substances and preparations (substances classified as carcinogens, mutagens or substances toxic to reproduction). L156/26

  20. Directive 2005/84/EC relating to restrictions on the marketing and use of certain dangerous substances and preparations (phthalates in toys and childcare articles). L 344/40

  21. Directive 2004/93/EC amending Council Directive 76/768/EEC for the purpose of adapting its Annexes II and III to technical progress. L 300/13

  22. Commission Regulation (EC) N) 552/2009 amending Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorization, and Restriction of Chemicals (REACH) as regards Annex XVII. 2009

  23. Frederiksen H, Jørgensen N, Andersson A-M. Correlations between phthalate metabolites in urine, serum, and seminal plasma from young Danish men determined by isotope dilution liquid chromatography tandem mass spectrometry. J Anal Toxicol. 2010;34:400–10.

    Article  CAS  Google Scholar 

  24. Huang P-C, Kuo P-L, Guo Y-L, Pao-Chi L, Ching-Chang L. Associations between urinary phthalate monoesters and thyroid hormones in pregnant women. Hum Reprod. 2007;22:2715–22.

    Article  CAS  Google Scholar 

  25. Preuss R, Koch HM, Angerer J. Biological monitoring of the five major metabolites of di-(2-ethylhexyl)phthalate (DEHP) in human urine using column-switching liquid chromatography-tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci. 2005;816:269–80.

    Article  CAS  Google Scholar 

  26. Kato K, Silva MJ, Wolf C, Gray LE, Needham LL, Calafat AM. Urinary metabolites of diisodecyl phthalate in rats. Toxicology. 2007;236:114–22.

    Article  CAS  Google Scholar 

  27. Kondo F, Ikai Y, Hayashi R, Okumura M, Takatori S, Nakazawua H, et al. Determination of five phthalate monoesters in human urine using gas chromatography-mass spectrometry. Bull Environ Contam Toxicol. 2010;85:92–6.

    Article  CAS  Google Scholar 

  28. Servaes K, Voorspoels S, Lievens J, Noten B, Allaerts K, Van De Weghe H, et al. Direct analysis of phthalate ester biomarkers in urine without preconcentration: method validation and monitoring. J Chromatogr A. 2013;1294:25–32.

    Article  CAS  Google Scholar 

  29. Chang YJ, Lin KL, Chang YZ. Determination of Di-(2 ethylhexyl)phthalate (DEHP) metabolites in human hair using liquid chromatography-tandem mass spectrometry. Clin Chim Acta. 2013;420:155–9.

    Article  CAS  Google Scholar 

  30. Hines EP, Calafat AM, Silva MJ, Mendola P, Fenton SE. Concentrations of phthalate metabolites in milk, urine, saliva, and serum of lactating North Carolina women. Environ Health Perspect. 2009;117:86–92.

    Article  CAS  Google Scholar 

  31. Silva MJ, Reidy JA, Samandar E, Herbert AR, Needham LL, Calafat AM. Detection of phthalate metabolites in human saliva. Arch Toxicol. 2005;79:647–52.

    Article  CAS  Google Scholar 

  32. Rezaee M, Assadi Y, Milani Hosseini M-R, Aghaee E, Ahmadi F, Berijani S. Determination of organic compounds in water using dispersive liquid–liquid microextraction. J Chromatogr A. 2006;1116:1–9.

    Article  CAS  Google Scholar 

  33. Han D, Row KH. Trends in liquid-phase microextraction and its application to environmental and biological samples. Microchim Acta. 2011;176:1–22.

    Article  Google Scholar 

  34. Alves ACH, Gonçalves MPB, Bernardo MMS, Mende B. Dispersive liquid–liquid microextraction of organophosphorous pesticides using nonhalogenated solvents. J Sep Sci. 2012;35:2653–8.

    Article  CAS  Google Scholar 

  35. Alves ACH, Gonçalves MPB, Bernardo MMS, Mende B. Validated dispersive liquid–liquid microextraction for analysis of organophosphorous pesticides in water. J Sep Sci. 2011;34:1326–32.

    Article  CAS  Google Scholar 

  36. Ghani JA, Choudhury IA. Application of Taguchi method in the optimization of end milling parameters. J Mater Process Technol. 2004;145:84–92.

    Article  CAS  Google Scholar 

  37. Albero B, Sánchez-Brunete C, García-Valcárcel AI, Pérez RA, Tadeo JL. Ultrasound-assisted extraction of emerging contaminants from environmental samples. Trends Anal Chem. 2015;71:110–8.

    Article  CAS  Google Scholar 

  38. Guo L, Lee HK. Vortex-assisted micro-solid-phase extraction followed by low-density solvent based dispersive liquid–liquid microextraction for the fast and efficient determination of phthalate esters in river water samples. J Chromatogr A. 2013;1300:24–30.

    Article  CAS  Google Scholar 

  39. Cinelli G, Avino P, Notardonato I, Centola A, Russo MV. Rapid analysis of six phthalate esters in wine by ultrasound-vortex-assisted dispersive liquid–liquid micro-extraction coupled with gas chromatography-flame ionization detector or gas chromatography-ion trap mass spectrometry. Anal Chim Acta. 2013;769:72–8.

    Article  CAS  Google Scholar 

  40. Pérez-Outeiral J, Millán E, Garcia-Arrona R. Determination of phthalate in food simulants and liquid samples using ultrasound-assisted dispersive liquid–liquid microextraction followed by solidification of floating organic drop. Food Control. 2016;62:171–7.

    Article  Google Scholar 

  41. Viñas P, Campillo N, Pastor-Belda P, Oller A, Hernández-Córdoba M. Determination of phthalate esters in cleaning and personal care products by dispersive liquid–liquid microextraction and liquid chromatography-tandem. J Chromatogr A. 2015;1376:18–25.

    Article  Google Scholar 

  42. Sun J-N, Shi Y-P, Chen J. Simultaneous determination of plasticizer di(2-ethylhexyl)phthalate and its metabolite in human urine by temperature controlled ionic liquid dispersive liquid–liquid microextraction combined with high performance liquid chromatography. Anal Methods. 2013;5:1427–34.

    Article  CAS  Google Scholar 

  43. Taguchi G, Yokoyama Y. Taguchi methods: design of experiments. Tokyo: American Supplier Institute, Dearborn MI: in conjunction with the Japanese Standards Association; 1994.

    Google Scholar 

  44. Munegumi T. Where is the border line between strong acids and weak acids? World J Chem Educ. 2013;1:12–6.

    Google Scholar 

Download references

Acknowledgments

The authors acknowledge the [European Union] Seventh Framework Programme ([FP7/2007-2013] under grant agreement no. [316665] (A-TEAM) by funding support of this research and PhD grant to A.A. (Marie Curie). The authors gratefully acknowledge the participants for the contribution of nail samples.

Author information

Authors and Affiliations

  1. Flemish Institute for Technological Research (VITO NV), Boeretang 200, 2400, Mol, Belgium

    Andreia Alves, Guido Vanermen & Stefan Voorspoels

  2. Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium

    Andreia Alves & Adrian Covaci

Authors
  1. Andreia Alves
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guido Vanermen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adrian Covaci
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Voorspoels
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Voorspoels.

Ethics declarations

All procedures performed in the present study were in accordance with the ethical standards of our institute. This study was approved by the Medical Ethics Committee in Antwerp University (Ethical approval register N.° B300201316329).

Conflict of interest

The authors declare that they have no conflict of interest.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 61 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Alves, A., Vanermen, G., Covaci, A. et al. Ultrasound assisted extraction combined with dispersive liquid–liquid microextraction (US-DLLME)—a fast new approach to measure phthalate metabolites in nails. Anal Bioanal Chem 408, 6169–6180 (2016). https://doi.org/10.1007/s00216-016-9727-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-016-9727-1

Keywords

Search

Navigation