Skip to main content
SpringerLink
Log in

Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt

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

Abstract

Within this study, a new method enabling monitoring of various estrogenic substances potentially occurring in milk and dairy products was proposed. Groups of compounds fairly differing in physico-chemical properties and biological activity were analyzed: four natural estrogens, four synthetic estrogens, five mycoestrogens, and nine phytoestrogens. Since they may pass into milk mainly in glucuronated and sulfated forms, an enzymatic hydrolysis was involved prior to the extraction based on the QuEChERS methodology. For the purification of the organic extract, a dispersive solid-phase extraction (d-SPE) with sorbent C18 was applied. The final analysis was performed by ultra-high-performance liquid chromatography (UHPLC) coupled with triple quadrupole tandem mass spectrometry (MS/MS). Method recovery ranged from 70 to 120% with a relative standard deviation (RSD) value lower than 20% and limits of quantification (LOQs) in the range of 0.02–0.60 μg/L (0.2–6.0 μg/kg dry weight) and 0.02–0.90 μg/kg (0.2–6.0 μg/kg dry weight) for milk and yogurt, respectively. The new procedure was applied for the investigation of estrogenic compounds in 11 milk samples and 13 yogurt samples from a Czech retail market. Mainly phytoestrogens were found in the studied samples. The most abundant compounds were equol and enterolactone representing 40–90% of all estrogens. The total content of phytoestrogens (free and bound) was in the range of 149–3870 μg/kg dry weight. This amount is approximately 20 times higher compared to non-bound estrogens.

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

Similar content being viewed by others

References

  1. Albertazzi P, Purdie DW. The nature and utility of the phytoestrogens: a review of the evidence. Maturitas. 2002;42:173–85.

    Article  CAS  Google Scholar 

  2. Fritsche S, Steinhart H. Occurrence of hormonally active compounds in food: a review. Eur Food Res Technol. 1999;209:153–79.

    Article  CAS  Google Scholar 

  3. EFSA Scientific Committee. Scientific opinion on the hazard assessment of endocrine disruptors: scientific criteria for identification of endocrine disruptors and appropriateness of existing test methods for assessing effects mediated by these substances on human health and the environment. EFSA J. 2013;11:3132. doi:10.2903/j.efsa.2013.3132.

    Article  Google Scholar 

  4. Gibson DA, Saunders PT. Endocrine disruption of oestrogen action and female reproductive tract cancers. Endocr Relat Cancer. 2014;21:T13–31.

    Article  CAS  Google Scholar 

  5. Malekinejad H, Scherpenisse P, Bergwerff AA. Naturally occurring estrogens in processed milk and in raw milk (from gestated cows). J Agric Food Chem. 2006;54:9785–91.

    Article  CAS  Google Scholar 

  6. Tso J, Aga DS. A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk. J Chrom A. 2010;1217:4784–95.

    Article  CAS  Google Scholar 

  7. Giusti MR, Iwamoto, Hatch EE. Diethylstilbestrol revisited: a review of the long-term health effects. Ann Intern Med. 1995;122:778–88.

    Article  CAS  Google Scholar 

  8. Council Directive 96/23/EC. Off J Eur Comm. 1996;L125(1996):10.

    Google Scholar 

  9. Kalac P. Fresh and ensiled forages as a source of estrogenic equol in bovine milk: a review. Czech J Anim Sci. 2013;58:296–303.

    CAS  Google Scholar 

  10. Höjer A, Adler S, Purup S, Hansen-Møller J, Martinsson K, Steinshamn H, Gustavsson AM. Effects of feeding dairy cows different legume-grass silages on milk phytoestrogen concentration. J Dairy Sci. 2012;95:4526–40.

    Article  Google Scholar 

  11. Njåstad KM, Adler SA, Hansen-Møller J, Thuen E, Gustavsson AM, Steinshamn H. Gastrointestinal metabolism of phytoestrogens in lactating dairy cows fed silages with different botanical composition. J Dairy Sci. 2014;97:7735–50.

    Article  Google Scholar 

  12. Daems F, Romnee JM, Heuskin S, Froidmont É, Lognay G. Analytical methods used to quantify isoflavones in cow’s milk: a review. Dairy Sci Technol. 2016;96:261–83.

    Article  CAS  Google Scholar 

  13. Wocławek-Potocka I, Mannelli C, Boruszewska D, Kowalczyk-Zieba I, Waśniewski T, Skarżyński DJ. Diverse effects of phytoestrogens on the reproductive performance: cow as a model. Int J Endocrinol. 2013;2013:1–15.

    Article  Google Scholar 

  14. Becker-Algeri TA, Castagnaro D, de Bortoli K, de Souza C, Drunkler DA, Badiale-Furlong E. Mycotoxins in bovine milk and dairy products: a review. J Food Sci. 2016;81:544–52.

    Article  Google Scholar 

  15. Kalac P. The effects of silage feeding on some sensory and health attributes of cow’s milk: a review. Food Chem. 2011;125:307–17.

    Article  CAS  Google Scholar 

  16. Meucci V, Soldani G, Razzuoli E, Saggese G, Massart F. Mycoestrogen pollution of Italian infant food. J Pediatr. 2011;159:278–83.

    Article  CAS  Google Scholar 

  17. Signorini ML, Gaggiotti M, Molineri A, Chiericatti CA, Zapata de Basílico ML, Basílico JC, Pisani M. Exposure assessment of mycotoxins in cow’s milk in Argentina. Food Chem Toxicol. 2012;50:250–7.

    Article  CAS  Google Scholar 

  18. Huang LC, Zheng N, Zheng BQ, Wen F, Cheng JB, Han RW, Xu XM, Li SL, Wang JQ. Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zearalenol in milk by UHPLC–MS/MS. Food Chem. 2014;146:242–9.

    Article  CAS  Google Scholar 

  19. Adamusova H, Bosakova Z, Coufal P, Pacakova V. Analysis of estrogens and estrogen mimics in edible matrices—a review. J Sep Sci. 2014;37:885–905.

    Article  CAS  Google Scholar 

  20. Capriotti AL, Cavaliere C, Colapicchioni V, Piovesana S, Samperi R, Laganà A. Analytical strategies based on chromatography–mass spectrometry for the determination of estrogen-mimicking compounds in food. J Chrom A. 2013;1313:62–77.

    Article  CAS  Google Scholar 

  21. Socas-Rodríguez B, Asensio-Ramos M, Hernández-Borges J, Herrera-Herrera AV, Rodríguez-Delgado M. Chromatographic analysis of natural and synthetic estrogens in milk and dairy products. TrAC Trend Anal Chem. 2013;44:58–77.

  22. Capriotti AL, Cavaliere C, Foglia P, Samperi R, Stampachiacchiere S, Ventura S, Laganà A. Ultra-high-performance liquid chromatography-tandem mass spectrometry for the analysis of free and conjugated natural estrogens in cow milk without deconjugation. Anal Bioanal Chem. 2015;407:1705–19.

    Article  CAS  Google Scholar 

  23. Cavaliere C, Capriotti AL, Foglia P, Piovesana S, Samperi R, Ventura S, Laganà A. Natural estrogens in dairy products: determination of free and conjugated forms by ultra high performance liquid chromatography with tandem mass spectrometry. J Sep Sci. 2015;38:3599–606.

    Article  CAS  Google Scholar 

  24. Capriotti AL, Cavaliere C, Piovesana S, Stampachiacchiere S, Samperi R, Ventura S, Laganà A. Simultaneous determination of naturally occurring estrogens and mycoestrogens in milk by ultrahigh-performance liquid chromatography–tandem mass spectrometry analysis. J Agric Food Chem. 2015;63:8940–6.

    Article  CAS  Google Scholar 

  25. D’Orazio G, Asensio-Ramos M, Hernández-Borges J, Rodríguez-Delgado M, Fanali S. Evaluation of the combination of a dispersive liquid–liquid microextraction method with micellar electrokinetic chromatography coupled to mass spectrometry for the determination of estrogenic compounds in milk and yogurt. Electrophoresis. 2015;36:615–25.

  26. Wielogórska E, Elliott CT, Danaher O, Chevallier O, Connolly L. Validation of an ultra high performance liquid chromatography–tandem mass spectrometry method for detection and quantitation of 19 endocrine disruptors in milk. Food Control. 2015;48:48–55.

    Article  Google Scholar 

  27. Anastassiades M, Lehotay SJ, Štajnbaher D, Schenck FJ. Fast and easy multiresidue method employing acetonitrile extraction/partitioning and dispersive solid-phase extraction for the determination of pesticide residues in produce. J AOAC Int. 2003;86:412–31.

    CAS  Google Scholar 

  28. Ehling S, Reddy TM. Liquid chromatography–mass spectrometry method for the quantitative determination of residues of selected veterinary hormones in powdered ingredients derived from bovine milk. J Agric Food Chem. 2013;61:11782–91.

    Article  CAS  Google Scholar 

  29. ČSN ISO 6731 (2011). Milk, cream and evaporated milk—determination of total solids content (reference method). This standard is the Czech version of the International Standard ISO 6731:2010.

  30. ČSN ISO 13580 (2007). Yogurt—determination of total solids content (reference method). This standard is the Czech version of International Standard ISO 13580:2005.

  31. Lankova D, Lacina O, Pulkrabova J, Hajslova J. The determination of perfluoroalkyl substances, brominated flame retardants and their metabolites in human breast milk and infant formula. Talanta. 2013;117:318–25.

    Article  CAS  Google Scholar 

  32. Sapozhnikova Y, Lehotay SJ. Multi-class, multi-residue analysis of pesticides, polychlorinated biphenyls, polycyclic aromatic hydrocarbons, polybrominated diphenyl ethers and novel flame retardants in fish using fast, low-pressure gas chromatography–tandem mass spectrometry. Anal Chim Acta. 2013;758:80–92.

    Article  CAS  Google Scholar 

  33. Rajski Ł, Lozano A, Uclés A, Ferrer C, Fernández-Alba AR. Determination of pesticide residues in high oil vegetal commodities by using various multi-residue methods and clean-ups followed by liquid chromatography tandem mass spectrometry. J Chromatogr A. 2013;1304:109–20.

  34. Lozano A, Rajski Ł, Uclés S, Belmonte-Valles N, Mezcua M, Fernández-Alba AR. Evaluation of zirconium dioxide-based sorbents to decrease the matrix effect in avocado and almond multiresidue pesticide analysis followed by gas chromatography tandem mass spectrometry. Talanta. 2014;118:68–83.

    Article  CAS  Google Scholar 

  35. Herrero P, Borrull F, Pocurull E, Marcé RM. A quick, easy, cheap, effective, rugged and safe extraction method followed by liquid chromatography-(Orbitrap) high resolution mass spectrometry to determine benzotriazole, benzothiazole and benzenesulfonamide derivates in sewage sludge. J Chromatogr A. 2014;1339:34–41.

  36. Rubert J, León N, Sáez C, Martins CPB, Godula M, Yusá V, Mañes J, Soriano JM, Soler C. Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry. Anal Chim Acta. 2014;820:39–46.

    Article  CAS  Google Scholar 

  37. Jia W, Chu X, Ling Y, Huang J, Chang J. Multi-mycotoxin analysis in dairy products by liquid chromatography coupled to quadrupole orbitrap mass spectrometry. J Chromatogr A. 2014;1345:107–14.

  38. Antignac JP, Cariou R, Le Bizec B, André F. New data regarding phytoestrogens content in bovine milk. Food Chem. 2004;87:275–81.

    Article  CAS  Google Scholar 

  39. Tsen SY, Siew J, EKL L, bte Roslee FA, Chan HM, Loke WM. Cow’s milk as a dietary source of equol and phenolic antioxidants: differential distribution in the milk aqueous and lipid fractions. Dairy Sci Technol. 2014;94:625–32.

    Article  CAS  Google Scholar 

  40. Hoikkala A, Mustonen E, Saastamoinen I, Jokela T, Taponen J, Saloniemi H, Wähälä K. High levels of equol in organic skimmed Finnish cow milk. Mol Nutr Food Res. 2007;51:782–6.

    Article  CAS  Google Scholar 

  41. Courant F, Antignac JP, Laille J, Monteau F, Andre F, Le Bizec B. Exposure assessment of prepubertal children to steroid endocrine disruptors. 2. Determination of steroid hormones in milk, egg, and meat samples. J Agric Food Chem. 2008;56:3176–84.

    Article  CAS  Google Scholar 

  42. Qin LQ, Wang PY, Kaneko T, Hoshi K, Sato A. Estrogen: one of the risk factors in milk for prostate cancer. Med Hypotheses. 2004;62:133–42.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

B.S.R. would like to thank the Canary Agency of Economy, Industry, Trade and Knowledge of the Government of the Canary Islands for the FPI fellowship (co-financed with 85% from European Social Funds). This work was supported by the Operational Programme Prague—Competitiveness (CZ.2.16/3.1.00/21537 and CZ.2.16/3.1.00/24503) and by the “National Program of Sustainability I”—NPU I (LO1215 and LO1601).

Author information

Authors and Affiliations

  1. Departamento de Química, Unidad Departamental de Química Analítica, Facultad de Ciencias, Universidad de La Laguna (ULL), Avenida Astrofísico Francisco Sánchez s/n°, 38206, San Cristóbal de La Laguna, Tenerife, Spain

    Bárbara Socas-Rodríguez, Javier Hernández-Borges & Miguel Ángel Rodríguez-Delgado

  2. Faculty of Food and Biochemical Technology, Department of Food Analysis and Nutrition, University of Chemistry and Technology, Prague, Technicka 3, 166 28, Prague, Czech Republic

    Bárbara Socas-Rodríguez, Darina Lanková, Kateřina Urbancová, Veronika Krtková, Jana Pulkrabová & Jana Hajšlová

Authors
  1. Bárbara Socas-Rodríguez
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Darina Lanková
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kateřina Urbancová
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Veronika Krtková
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Javier Hernández-Borges
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Miguel Ángel Rodríguez-Delgado
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jana Pulkrabová
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jana Hajšlová
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jana Hajšlová.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Electronic supplementary material

ESM 1

(PDF 184 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Socas-Rodríguez, B., Lanková, D., Urbancová, K. et al. Multiclass analytical method for the determination of natural/synthetic steroid hormones, phytoestrogens, and mycoestrogens in milk and yogurt. Anal Bioanal Chem 409, 4467–4477 (2017). https://doi.org/10.1007/s00216-017-0391-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0391-x

Keywords

Search

Navigation