Determination of Corticosteroids in Fish Plasma by High Performance Liquid Chromatography: Evaluation of the Method Using Striped Bass (Morone saxatilis)

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Abstract
A sensitive, specific, and reproducible technique for the simultaneous determination of cortisol, cortisone, and corticosterone in fish plasma was developed using high performance liquid chromatography (HPLC). Corticosteroids were extracted from plasma by methylene chloride and Sep-Pak cartridges for comparative purposes. The Sep-Pak method proved more convenient and efficient. Chromatography was accomplished on a nitrile bonded phase using a methanol–water mobile phase. Steroids were identified and quantified by monitoring absorbance at 254 and 280 nm simultaneously. Sensitivity is approximately 5 ng for each steroid. An evaluation of the technique using subadult striped bass (Morone saxatilis) revealed that cortisol was the only corticosteroid detected. Plasma cortisol concentrations increased in response to brief confinement and crowding, supporting the efficacy of this measurement as an evaluator of stress in striped bass. The HPLC technique provides specific determination of each corticosteroid (thus eliminating cross-reactivity problems inherent in radioimmunoassay methods), resolves corticosteroids from androgens and estrogens, and permits the isolation and purification of individual steroids.

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