Development and Validation of Urine Hormones by Lc-Ms/Ms Using the Boreal Toad (Anaxyrus Boreas Boreas) as a Model Species

Analysis of urinary hormone levels can be used to identify sex and breeding patterns in amphibians. This is especially important for ensuring the survival of endangered species. The most common way to determine hormone levels is enzyme-linked immunosorbent assay (ELISA); however, there is the possibility of cross-reactivity with this technique, and only one hormone can be monitored per plate, limiting efficiency. In this study, the effectiveness of liquid chromatography coupled to mass spectrometry (LC-MS/MS) was evaluated in terms of sensitivity, repeatability, and through-put in monitoring hormone levels in Anaxyrus boreas boreas (boreal toads). Urine samples were collected from ten different female Boreal toads over a three-year period and monitored for estrone, â-estradiol, estriol, 17á-ethinylestradiol, corticosterone, testosterone, and progesterone. To account for differences in urine concentration, creatinine (a metabolic breakdown product) was used to normalize all samples as it is typically present at a constant concentration. Prior to chromatographic analysis, all samples underwent a solid phase extraction (SPE) clean-up. Hormones in the estrogen class were derivatized via dansylation prior to analysis to enhance detection sensitivity. An Agilent 1290 Liquid Chromatograph coupled to an Agilent 6460 Triple Quadrupole Mass Spectrometer was used to quantitate hormone levels. The extraction method developed proved to be reliable and reproducible with percent recoveries in quality control samples ranging from 80%-120%. Using LC-MS/MS and derivatization, limits of quantitation as low as 0.001 parts-per-billion (ppb) were achieved for estrogens and 0.100 ppb for androgens. Ultimately, the method optimized for hormone analysis using LC-MS/MS showed to be as reliable as ELISA while also being less expensive per sample and more robust