Analytical and Bioanalytical Chemistry

, Volume 403, Issue 6, pp 1629–1640 | Cite as

Quantification of steroids and endocrine disrupting chemicals in rat ovaries by LC-MS/MS for reproductive toxicology assessment

  • Nadia QuignotEmail author
  • Mikaël Tournier
  • Charlène Pouech
  • Cécile Cren-Olivé
  • Robert Barouki
  • Emmanuel Lemazurier
Original Paper


Reproductive function is controlled by a finely tuned balance of androgens and estrogens. Environmental toxicants, notably endocrine disrupting chemicals (EDCs), appear to be involved in the disruption of hormonal balance in several studies. To further describe the effects of selected EDCs on steroid secretion in female rats, we aim to simultaneously investigate the EDC concentration and the sex hormone balance in the ovaries. Therefore, an effective method has been developed for the quantification of the sex steroid hormones (testosterone, androstenedione, estradiol, and estrone) and four endocrine disrupting chemicals (bisphenol A, atrazine, and the active metabolites of methoxychlor and vinclozolin) in rat ovaries. The sample preparation procedure is based on the so-called “quick, easy, cheap, effective, rugged, and safe” approach, and an analytical method was developed to quantify these compounds with low detection limits by liquid chromatography coupled with a tandem mass spectrometer. This analytical method, applied to rat ovary samples following subacute EDC exposure, revealed some new findings for toxicological evaluation. In particular, we showed that EDCs with the same described in vitro mechanisms of action have different effects on the gonadal steroid balance. These results highlight the need to develop an integrative evaluation with the simultaneous measurement of EDCs and numerous steroids for good risk assessment.


Ovaries from rats treated or not with model endocrine disrupting chemicals were subjected to LC-MS/MS analysis for the simultaneous quantification of chemicals and sex hormones.


Endocrine disrupting chemicals Hormonal balance LC-MS/MS Steroids Toxicity 



We would like to thank Franck Robidel and Anthony Lecomte for their help with animal care. We are grateful to Laure Wiest and Robert Baudot for their assistance using the mass spectrometry facilities. Last but not least, we thank Diana Z. and Andrew L. from American Journal Experts for their editorial assistance.


This project was supported by a grant from The French Environment Ministry and by Ph.D. training support from The National Institute of Industrial Environment and Risk (INERIS).

Conflict of interest

The authors declare no conflicts of interest.

Supplementary material

216_2012_5990_MOESM1_ESM.pdf (247 kb)
ESM 1 (PDF 246 kb)


  1. 1.
    Rogan WJ, Ragan NB (2007) Some evidence of effects of environmental chemicals on the endocrine system in children. Int J Hyg Environ Health 210(5):659–667CrossRefGoogle Scholar
  2. 2.
    Vinggaard AM, Hnida C, Breinholt V, Larsen JC (2000) Screening of selected pesticides for inhibition of CYP19 aromatase activity in vitro. Toxicol In Vitro 14(3):227–234CrossRefGoogle Scholar
  3. 3.
    Toppari J, Larsen JC, Christiansen P, Giwercman A, Grandjean P, Guillette LJ Jr, Jegou B, Jensen TK, Jouannet P, Keiding N, Leffers H, McLachlan JA, Meyer O, Muller J, Rajpert-De Meyts E, Scheike T, Sharpe R, Sumpter J, Skakkebaek NE (1996) Male reproductive health and environmental xenoestrogens. Environ Health Perspect 104(Suppl 4):741–803CrossRefGoogle Scholar
  4. 4.
    Hiroi H, Tsutsumi O, Momoeda M, Takai Y, Osuga Y, Taketani Y (1999) Differential interactions of bisphenol A and 17beta-estradiol with estrogen receptor alpha (ERalpha) and ERbeta. Endocr J 46(6):773–778CrossRefGoogle Scholar
  5. 5.
    Holloway AC, Anger DA, Crankshaw DJ, Wu M, Foster WG (2008) Atrazine-induced changes in aromatase activity in estrogen sensitive target tissues. J Appl Toxicol 28(3):260–270CrossRefGoogle Scholar
  6. 6.
    Molina-Molina JM, Hillenweck A, Jouanin I, Zalko D, Cravedi JP, Fernandez MF, Pillon A, Nicolas JC, Olea N, Balaguer P (2006) Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicol Appl Pharmacol 216(1):44–54CrossRefGoogle Scholar
  7. 7.
    Shelby MD, Newbold RR, Tully DB, Chae K, Davis VL (1996) Assessing environmental chemicals for estrogenicity using a combination of in vitro and in vivo assays. Environ Health Perspect 104(12):1296–1300CrossRefGoogle Scholar
  8. 8.
    Amato MC, Verghi M, Nucera M, Galluzzo A, Giordano C (2011) Low estradiol-to-testosterone ratio is associated with oligo-anovulatory cycles and atherogenic lipidic pattern in women with polycystic ovary syndrome. Gynecol Endocrinol 27(8):579–586CrossRefGoogle Scholar
  9. 9.
    Clode SA (2006) Assessment of in vivo assays for endocrine disruption. Best Pract Res Clin Endocrinol Metab 20(1):35–43CrossRefGoogle Scholar
  10. 10.
    Flores-Valverde AM, Hill EM (2008) Methodology for profiling the steroid metabolome in animal tissues using ultraperformance liquid chromatography-electrospray-time-of-flight mass spectrometry. Anal Chem 80(22):8771–8779CrossRefGoogle Scholar
  11. 11.
    Labadie P, Budzinski H (2006) Alteration of steroid hormone balance in juvenile turbot (Psetta maxima) exposed to nonylphenol, bisphenol A, tetrabromodiphenyl ether 47, diallylphthalate, oil, and oil spiked with alkylphenols. Arch Environ Contam Toxicol 50(4):552–561CrossRefGoogle Scholar
  12. 12.
    Strahm E, Kohler I, Rudaz S, Martel S, Carrupt PA, Veuthey JL, Saugy M, Saudan C (2008) Isolation and quantification by high-performance liquid chromatography-ion-trap mass spectrometry of androgen sulfoconjugates in human urine. J Chromatogr A 1196–1197:153–160CrossRefGoogle Scholar
  13. 13.
    Harwood DT, Handelsman DJ (2009) Development and validation of a sensitive liquid chromatography-tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization. Clin Chim Acta 409(1–2):78–84CrossRefGoogle Scholar
  14. 14.
    Vulliet E, Wiest L, Baudot R, Grenier-Loustalot MF (2008) Multi-residue analysis of steroids at sub-ng/l levels in surface and ground-waters using liquid chromatography coupled to tandem mass spectrometry. J Chromatogr A 1210(1):84–91CrossRefGoogle Scholar
  15. 15.
    Tso J, Aga DS (2010) A systematic investigation to optimize simultaneous extraction and liquid chromatography tandem mass spectrometry analysis of estrogens and their conjugated metabolites in milk. J Chromatogr A 1217(29):4784–4795CrossRefGoogle Scholar
  16. 16.
    Jantti SE, Tammimaki A, Raattamaa H, Piepponen P, Kostiainen R, Ketola RA (2010) Determination of steroids and their intact glucuronide conjugates in mouse brain by capillary liquid chromatography–tandem mass spectrometry. Anal Chem 82(8):3168–3175CrossRefGoogle Scholar
  17. 17.
    Stubbings G, Bigwood T (2009) The development and validation of a multiclass liquid chromatography tandem mass spectrometry (LC-MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS (quick, easy, cheap, effective, rugged and safe) approach. Anal Chim Acta 637(1–2):68–78CrossRefGoogle Scholar
  18. 18.
    Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ (2003) 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 86(2):412–431Google Scholar
  19. 19.
    Zhao L, Stevens J (2010) Determination of quinolone antibiotics in bovine liver using Agilent SampliQ QuEChERs kits by LC/MS/MS. 2010 edn. Agilent Technologies, Inc., WilmingtonGoogle Scholar
  20. 20.
    Costain RM, Fesser AC, McKenzie D, Mizuno M, MacNeil JD (2008) Identification of hormone esters in injection site in muscle tissues by LC/MS/MS. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 25(12):1520–1529CrossRefGoogle Scholar
  21. 21.
    Xu J, Wu L, Chen W, Chang AC (2008) Simultaneous determination of pharmaceuticals, endocrine disrupting compounds and hormone in soils by gas chromatography-mass spectrometry. J Chromatogr A 1202(2):189–195CrossRefGoogle Scholar
  22. 22.
    Regal P, Vazquez BI, Franco CM, Cepeda A, Fente C (2009) Quantitative LC-MS/MS method for the sensitive and simultaneous determination of natural hormones in bovine serum. J Chromatogr B Analyt Technol Biomed Life Sci 877(24):2457–2464CrossRefGoogle Scholar
  23. 23.
    Harvey CN, Esmail M, Wang Q, Brooks AI, Zachow R, Uzumcu M (2009) Effect of the methoxychlor metabolite HPTE on the rat ovarian granulosa cell transcriptome in vitro. Toxicol Sci 110(1):95–106CrossRefGoogle Scholar
  24. 24.
    Sierra-Santoyo A, Castaneda-Hernandez G, Harrison RA, Barton HA, Hughes MF (2008) Pharmacokinetics and dosimetry of the antiandrogen vinclozolin after oral administration in the rat. Toxicol Sci 106(1):55–63CrossRefGoogle Scholar
  25. 25.
    Okazaki K, Okazaki S, Nishimura S, Nakamura H, Kitamura Y, Hatayama K, Nakamura A, Tsuda T, Katsumata T, Nishikawa A, Hirose M (2001) A repeated 28-day oral dose toxicity study of methoxychlor in rats, based on the ‘enhanced OECD test guideline 407’ for screening endocrine-disrupting chemicals. Arch Toxicol 75(9):513–521CrossRefGoogle Scholar
  26. 26.
    Shibayama H, Kotera T, Shinoda Y, Hanada T, Kajihara T, Ueda M, Tamura H, Ishibashi S, Yamashita Y, Ochi S (2009) Collaborative work on evaluation of ovarian toxicity. 14) Two- or four-week repeated-dose studies and fertility study of atrazine in female rats. J Toxicol Sci 34(Suppl 1):SP147–SP155CrossRefGoogle Scholar
  27. 27.
    Shin JH, Moon HJ, Kim TS, Kang IH, Ki HY, Choi KS, Han SY (2006) Repeated 28-day oral toxicity study of vinclozolin in rats based on the draft protocol for the “Enhanced OECD Test Guideline No. 407” to detect endocrine effects. Arch Toxicol 80(9):547–554CrossRefGoogle Scholar
  28. 28.
    Quignot N, Arnaud M, Robidel F, Lecomte A, Tournier M, Cren-Olive C, Barouki R, Lemazurier E (2012) Characterization of endocrine-disrupting chemicals based on hormonal balance disruption in male and female adult rats. Reprod Toxicol.
  29. 29.
    Goldman JM, Murr AS, Cooper RL (2007) The rodent estrous cycle: characterization of vaginal cytology and its utility in toxicological studies. Birth Defects Res B Dev Reprod Toxicol 80(2):84–97CrossRefGoogle Scholar
  30. 30.
    ICH (2005) Paper presented at the ICH harmonised tripartite guideline, validation of analytical procedures: text and methodology Q2(R1). Paper presented at the International Conference on Harmonisation of technical requirements for registration of pharmaceuticals for human use, GenevaGoogle Scholar
  31. 31.
    Mansilha C, Melo A, Rebelo H, Ferreira IM, Pinho O, Domingues V, Pinho C, Gameiro P (2010) Quantification of endocrine disruptors and pesticides in water by gas chromatography–tandem mass spectrometry. Method validation using weighted linear regression schemes. J Chromatogr A 1217(43):6681–6691CrossRefGoogle Scholar
  32. 32.
    Hewavitharana AK (2011) Matrix matching in liquid chromatography-mass spectrometry with stable isotope labelled internal standards—is it necessary? J Chromatogr A 1218(2):359–361CrossRefGoogle Scholar
  33. 33.
    Majors RE (2008) QuEChERS—a new technique for multiresidue analysis of pesticides in foods and agricultural samples. Lc Gc Asia Pac 11Google Scholar
  34. 34.
    Przybylski C, Segard C (2009) Method for routine screening of pesticides and metabolites in meat based baby-food using extraction and gas chromatography–mass spectrometry. J Sep Sci 32(11):1858–1867CrossRefGoogle Scholar
  35. 35.
    Manson JE (2008) Prenatal exposure to sex steroid hormones and behavioral/cognitive outcomes. Metabolism 57(Suppl 2):S16–S21CrossRefGoogle Scholar
  36. 36.
    Stoker TE, Laws SC, Guidici DL, Cooper RL (2000) The effect of atrazine on puberty in male Wistar rats: an evaluation in the protocol for the assessment of pubertal development and thyroid function. Toxicol Sci 58(1):50–59CrossRefGoogle Scholar
  37. 37.
    Sanderson JT, Boerma J, Lansbergen GW, van den Berg M (2002) Induction and inhibition of aromatase (CYP19) activity by various classes of pesticides in H295R human adrenocortical carcinoma cells. Toxicol Appl Pharmacol 182(1):44–54CrossRefGoogle Scholar
  38. 38.
    Akgul Y, Derk RC, Meighan T, Rao KM, Murono EP (2008) The methoxychlor metabolite, HPTE, directly inhibits the catalytic activity of cholesterol side-chain cleavage (P450scc) in cultured rat ovarian cells. Reprod toxicol (Elmsford, NY) 25(1):67–75CrossRefGoogle Scholar
  39. 39.
    Quignot N, Desmots S, Barouki R, Lemazurier E (2012) A comparison of two human cell lines and two rat gonadal cell primary cultures as in vitro screening tools for aromatase modulation. Toxicol In Vitro 26(1):107–118CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Nadia Quignot
    • 1
    Email author
  • Mikaël Tournier
    • 2
  • Charlène Pouech
    • 2
  • Cécile Cren-Olivé
    • 2
  • Robert Barouki
    • 3
  • Emmanuel Lemazurier
    • 1
  1. 1.Experimental Toxicology UnitINERIS, Parc Technologique ALATAVerneuil-en-HalatteFrance
  2. 2.Département Service Central d’AnalyseUMR CNRS 5280, Institut des Sciences AnalytiquesSolaizeFrance
  3. 3.INSERM UMR-S 747, Université Paris DescartesParisFrance

Personalised recommendations