Development of a protocol to profile the gonadal steroid intermediates of vitellogenic ovaries from fathead minnows, pimephales promelas

Abstract

This research developed a protocol to extract, isolate, detect, and measure the intermediates involved in ovarian steroid biosynthesis by Pimephales promelas to be used in bleached kraft mill effluent chronic toxicity studies. Steroid extraction from ovarian tissue was accomplished by sonication in methanol with 1:1 tissue:solvent. Four such extractions per sample successfully increased percent recovery from <58% to >74%, depending on the steroid. Isolation of steroids from the sample matrix was accomplished by C18 solid phase extraction columns. Adjustment of sample matrix pH to 6.5, and polarity of eluant to 7:3 hexane/t-butyl ether successfully removed free fatty acid interferences from the chromatographic analysis. The isolated steroids were derivatized to produce methoxime-trimethylsilyl derivatives that were detected and measured using selected ion recording - gas chromatography - mass spectroscopy. The finalized protocol had a percent recovery ranging from 74% to 101%, and a reproducibility of <11%, depending on the steroid. Detection limit standard curves were linear (0.9740<r<0.9994, depending on steroid).