HPLC Applications

The Wisconsin Regional Primate Research Center provides HPLC separation and analysis on a charge basis. The HPLC system currently consists of Beckman HPLC pumps, automated sampler, UV diode array detector, fractionator and computer run analysis through System Gold Nouveau software. The system is used for validation of hormones analyzed by assay systems, identification, and quantification. Methods for separating, identifying and quantifying steroids are currently available.

Recent methods for quantifying androgen metabolites and glucocorticoids in biological samples have been developed. Here's a chromatograph showing the current steroids measured. These nine steroids and internal standard are measured in individual samples during a forty minute run.

Validations have been performed for measuring male rhesus androgen metabolites and adrenal glucocorticoids in urine. Chromatographs of intact and castrated male urine demonstrate the gonadal androgen metabolites to be measured.

Steroids absorb light at different wavelenghts. Optimal sensitivity can be determined for each steroid by finding the optimal wavelenght. This 3D chromatograph illustrates steroid UV absorption.

Methods for separating and quantifying protein hormones are underway. The assay labs will also provide consultation and training on the system in certain cases.
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