Solid-phase Extraction of Estradiol-17β from Bovine Blood Plasma for its Peripheral Detection by Radioimmunoassay

Abstract

A solid-phase extraction procedure for estradiol-17β from bovine blood plasma with a reversed phase cartridge was developed for the measurement of its concentration by radioimmunoassay. Estradiol-17β retained in the cartridge was eluted quantitatively with 70%(v/v) methanol, although estrone was recovered in the same fraction. The apparent values measured by dextran-coated charcoal radioimmunoassay with an eluent of 75%(v/v) methanol in the solid-phase extraction declined approximately by 10% as applied plasma volume was increased by 1 ml (r=-0.98, p<0.001). However, the values by double antibody radioimmunoassay were elevated approximately 8% per 1 ml plasma increase (r=0.82, p<0.001). The recovery of estradiol-17β added to bovine plasma in the extraction and radioimmunoassay with dextran-coated charcoal separation was approximately 75% under conditions of 2 ml plasma application, and concentrations calculated using buffer standards. Matrix effects on the assay recovery were negligible in the addition of solid-phase extract of castrated bull plasma to the standards. On the other hand, the average recoveries calculated with buffer and process standards in double antibody radioimmunoassay were 170% and 110%, respectively. The assay value in the solid-phase extraction showed good agreement with the value in the liquid-liquid extraction and liquid chromatography after correction with the recovery rate (r=0.99). In conclusion, solid-phase extraction with a reversed phase cartridge is a practical extraction method for the measurement of estradiol-17β during the estrous cycle in cattle.