title: Two simple cleanup methods combined with LC-MS/MS for quantification of steroid hormones in in vivo and in vitro assays.
abstract: Measuring both progestagens, androgens, corticosteroids as well as estrogens with a single method makes it possible to investigate the effects of endocrine-disrupting chemicals (EDCs) on the main pathways in the mammalian steroidogenesis. This paper presents two simple methods for the determination of the major steroid hormones in biological matrixes using liquid chromatography tandem mass spectrometry (LC-MS(2)). A novel method was developed for the determination of 14 steroids in the H295R in vitro assay without the need for solid phase extraction (SPE) purification prior to LC-MS(2) analysis. The in vitro assay was validated by exposing H295R cells to prochloraz for inhibiting steroid hormone secretion and by exposing cells to forskolin for inducing steroid hormone secretion. The developed method fulfills the recommendations for the H295R assay suggested by the OECD. Furthermore, a simple off-line SPE methodology was developed for the necessary clean-up of in vivo assays. Samples, such as gonad tissue, plasma and serum, are complex biological matrixes, and the SPE methodology was optimized to remove salts and proteins prior to elution of target analytes. At the same time, lipophilic compounds were retained on the SPE cartridge during elution. This, combined with the multi-steroid LC-MS(2) method, made it possible to determine 10 steroids in male Sprague-Dawley rat gonad tissue. Furthermore, it was possible to quantify 6 steroids in the plasma. In general, the observed concentration of steroid hormones in plasma, testes, and H295R cell medium corresponded well with previous studies. The off-line SPE method was validated using spiked charcoal-stripped serum. Method recovery, accuracy, precision and robustness were all good. Instrument sensitivity was in the range of 55-530 pg/mL (LLOQ).
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Johan Juhl
authors.initials: JJ
authors.lastname: Weisser
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Cecilie Hurup
authors.initials: CH
authors.lastname: Hansen
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Rikke
authors.initials: R
authors.lastname: Poulsen
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Lizette Weber
authors.initials: LW
authors.lastname: Larsen
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark.
authors.forename: Claus
authors.initials: C
authors.lastname: Cornett
authors.affiliation: Toxicology Laboratory, Analytical BioSciences, Department of Pharmacy, Faculty of Health and Medical Science, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark. bjarne.styrishave@sund.ku.dk.
authors.forename: Bjarne
authors.initials: B
authors.lastname: Styrishave
mesh_terms: D000818:Animals; D001681:Biological Assay / Q000379:methods*; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods*; D052244:Endocrine Disruptors / Q000008:administration & dosage*; D012739:Gonadal Steroid Hormones / Q000097:blood / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013048:Specimen Handling / Q000379:methods*; D013737:Testis / Q000187:drug effects / Q000378:metabolism*
chemical_list: D052244:Endocrine Disruptors; D012739:Gonadal Steroid Hormones
keywords: Gonads; H295R cell assay; Online cleanup; Plasma; Solid phase extraction; Testis