Development of a highly sensitive and specific assay for plasma ethinylestradiol using combined extraction, liquid chromatography and radioimmunoassay

Abstract

A highly sensitive and specific assay for ethinylestradiol (EE2) in human plasma was developed. The assay procedure combined solid-phase extraction of plasma samples, isolation of extracted EE2 by liquid chromatography (LC), and radioimmunoassay. Samples were extracted to remove polar plasma constituents and steroid binding proteins. Chromatography was employed to separate EE2 from other steroids that were candidates for assay cross-reactivity. The radioimmunoassay was shown to be sensitive (lower limit of quantitation = 2 pg ml-1 EE2 in plasma) and accurate (mean accuracy = 102%). Recovery of EE2 through extraction and LC steps was 76.1 +/- 4.5% (mean +/- SD; n = 42). Overall assay intra- and inter-assay coefficients of variation were 3.6 and 8.9%, respectively. The analyte was stable in assay buffer and assay accuracy was influenced minimally by four sample freeze-thaw cycles. This assay protocol enables the precise monitoring of low circulating levels of EE2, a prominent and potent synthetic oestrogen.