| dc.description.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). | |
