title: Measurement of steroids in rats after exposure to an endocrine disruptor: mass spectrometry and radioimmunoassay demonstrate similar results.
abstract: BACKGROUND
  Commercially available radioimmunoassays (RIAs) are frequently used to evaluate the effects of endocrine disrupting chemicals (EDCs) on steroidogenesis in rats. Currently there are limited data comparing steroid concentrations in rats as measured by RIAs to those obtained using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This study evaluates the concordance of serum and urine steroid concentrations as quantified by select RIA kits and LC-MS/MS following exposure to an EDC, atrazine (ATR).
  
  
  METHODS
  Adult male rats were orally dosed with ATR (200 mg/kg/day) or methylcellulose (1%, vehicle control) for 5 days. Serum was collected and separated into aliquots for analysis. Serum was assayed by RIA for androstenedione (ANDRO), corticosterone (CORT), estradiol (E2), estrone (E1), progesterone (P4), and testosterone (T). Serum was extracted prior to LC-MS/MS analysis with positive electrospray ionization in multiple-reaction monitoring mode for ANDRO, CORT, P4, and T. E1 and E2 concentrations were quantified similarly by LC-MS/MS, following derivatization with dansyl chloride. To compare CORT values from urine, pregnant adult rats were orally dosed with either ATR (100 mg/kg/day) or methylcellulose for 5 days (i.e., gestational days 14-18). Urine samples were collected daily and assayed for CORT by RIA and LC-MS/MS as described above.
  
  
  RESULTS
  Data analyses demonstrated significant agreement between the two detection methods as assessed by Pearson product-moment correlation coefficient, Bland-Altman analysis, and the interclass correlation coefficient. No statistically significant differences were observed between RIA and LC-MS/MS means for any of the steroids assayed.
  
  
  CONCLUSIONS
  These findings indicate a significant correlation between the measurement of steroids within rat serum and urine using RIA kits and LC-MS/MS. Differences in the absolute measurements existed, but these were not statistically significant. These findings indicate that steroids may be reliably measured in rat biological media using RIAs or LC-MS/MS.
authors.affiliation: Oak Ridge Institute for Science and Education (ORISE), Research Participation Program, Oak Ridge, TN 37831, United States; Endocrine Toxicology Branch, Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory (NHEERL), Office of Research and Development (ORD), U.S. Environmental Protection Agency (U.S. EPA), Research Triangle Park, NC 27711, United States.
authors.forename: Brandy W
authors.initials: BW
authors.lastname: Riffle
authors.forename: W Matthew
authors.initials: WM
authors.lastname: Henderson
authors.forename: Susan C
authors.initials: SC
authors.lastname: Laws
mesh_terms: D000284:Administration, Oral; D000818:Animals; D001280:Atrazine / Q000633:toxicity*; D002853:Chromatography, Liquid / Q000379:methods*; D003345:Corticosterone / Q000097:blood / Q000652:urine; D052244:Endocrine Disruptors / Q000633:toxicity*; D008297:Male; D011863:Radioimmunoassay / Q000379:methods*; D051381:Rats; D017208:Rats, Wistar; D015203:Reproducibility of Results; D013256:Steroids / Q000097:blood / Q000652:urine; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D052244:Endocrine Disruptors; D013256:Steroids; D001280:Atrazine; D003345:Corticosterone
keywords: ANDRO; ANOVA; ATR; AUC; CORT; CV; E1; E2; EDC; FIFRA SAP; Federal Insecticide, Fungicide, and Rodenticide Act Scientific Advisory Panel; GD; GLM; HPA; ICC; LC–MS/MS; LH; MRM; Mass spectroscopy; Methods; P4; Quality control; RIA; Radioimmunoassay; SD; SEM; SPE; SRM; Steroids; T; analysis of variance; androstenedione; area under the curve; atrazine; coefficient of variation; corticosterone; endocrine disrupting chemical; estradiol; estrone; general linear model; gestational day; hypothalamic–pituitary–adrenal; intraclass correlation coefficients; liquid chromatography coupled with tandem mass spectroscopy; luteinizing hormone; multiple reaction monitoring mode; progesterone; radioimmunoassay; selective reaction monitoring; solid phase extraction; standard deviation; standard error mean; testosterone