Florida Atlantic University, Department of Biological Sciences, Schmidt College of Science, Boca Raton, FL, USA
Abstract
Serum and urine samples from Tursiops truncatus (Atlantic bottlenose dolphins), obtained from animals in the Indian River Lagoon and Charleston Harbor as part of the Dolphin Health and Risk Assessment study (HERA), were analyzed for steroid concentrations by preparative reversed-phase performance liquid chromatography (RP-HPLC). Comparison assays were performed on green iguanas and red-eared sliders (turtles). The latter species are both considered invasive in Florida, and are used as a model invasive species and for model hypoxia studies respectively. Steroids of interest were estradiol (E2), estrone (E1), estriol (E3), testosterone (T2), and progesterone (P4).1,2 Separation was performed with a Waters® C18 column was used on a Hitachi® EZ Chrome® HPLC. Fifteen minute isocratic and gradient elutions were run with water and acetonitrile.3,4 Additional filtering of solvents and sample were performed just upstream of the column with an All-Tech® guard column and cartridge assembly. Samples were extracted in alcohol by a multi-step extraction and separation procedure. Standards mixtures were developed with pure lyophilized steroid at four concentrations, and recoveries were determined by extracting standards by the same means as the samples. Following preliminary assays, retention times were determined, and separated, purified steroids were obtained from samples during HPLC runs to be used in Western Blot and HPLC/electron spray mass spectrometry (HPLC/ESI-MS-MS). These assays permit ready confirmation of the relative concentrations of the steroids and receptors of interest, and provide a simple confirmation of concurrent enzyme-linked immunosorbent assay (ELISA) results, as well as a means of further purifying steroids for additional analysis.
Acknowledgements
Grateful acknowledgement is extended to Dr. Gregory Bossart, Dr. Patricia Fair and Dr. Sarah Milton for support and access to samples and data, and to Ms. Ashley Campbell, Mr. George Biedenbach and Mr. Mike Denney for samples. Mr. Chris Hall and Mr Jim Nichol from Hitachi have been invaluable resources for all things HPLC. Financial support has been provided by the FAU National Alumni Association, the FAU Graduate Student Association, FAU Student Financial Aid scholarships, the Department of Biological Sciences, and the Capt. Nathan Award.
References
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2. Kessler MJ. High performance liquid chromatography of steroid metabolites in the pregnenolone and progesterone pathways. Steroids 1982; 39(1):21–32.
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