Semester

Spring

Date of Graduation

2009

Document Type

Dissertation

Degree Type

PhD

College

Eberly College of Arts and Sciences

Department

Chemistry

Committee Chair

Lisa A Holland

Abstract

This dissertation is based on research that led to the development and application of capillary electrophoresis method for the analysis of sex steroid hormones in the blood or plasma of fish species. The analysis of steroid hormones is essential to reveal chemical compounds that are suspected in endocrine disruption, but it is challenging due to the similarity of the chemical structures of steroids, their low concentrations, and limited volumes of plasma or blood samples in fish available for analysis. There is therefore an acute need to develop reliable accurate and systematic analytical methods applicable for the analysis of structurally similar steroid hormones at very low concentrations. The method developed here is based on micellar electrokinetic chromatography, a type of capillary electrophoresis that incorporates secondary equilibria. The method utilizes pH-stacking for steroid preconcentration to improve the detection limits. This method of pH-stacking is accomplished by using charged derivatives of cyclodextrin which become neutral (protonated) or anionic (deprotonated) based on the pH of the sample buffer. Preconcentration by means of pH-stacking occurs upon introduction of the sample into the capillary at the pH junction, resulting in a fast and efficient separation analysis of steroids. Using the developed method the separation of eight targeted steroids, that include alpha,beta-dihydroxyprogesterone, ethynylestradiol, 17beta-estradiol, estrone, hydroxyprogesterone, 11-ketotestosterone, progesterone, and testosterone is achieved in less than 4 min which is substantially faster than steroid analysis by immunoassay, GC-MS or LC-MS. For all targeted steroids, the within-day and day-to-day reproducibility in migration time is <1 and <2% relative standard deviation (RSD), respectively. The reproducibility in peak area obtained in aqueous samples is below 6% and 22% (RSD) within-day and day-to-day respectively. The limits of detection range from 2 to 14 nM using a 60 s electrokinetic injection. The method is validated by measuring the recovery of standard steroids added to the aqueous or fish plasma samples prior to sample preparation. The recovery of testosterone and 17beta-estradiol added to the fish plasma prior to sample preparation range from 74% to 102%. The method is successfully applied to the determination of sex steroid levels in blood plasma of yellow perch captured from natural aqueous habitats. The results are compared to radioimmunoassay.

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