A simple, differential extraction method for the simultaneous direct radioimmunoassay of androgens and androgen glucuronides in human serum.

A method is described for the differential extraction of unconjugated androgens (testosterone and dihydrotestosterone) and glucuronidated androgens (androstane-3 alpha,17 beta-diol glucuronide and androsterone glucuronide) from human serum using solid-phase, gravity-flow extraction columns. In this method, 100-microL aliquots of serum are loaded onto the normal-phase columns, unconjugated androgens are eluted with ethyl ether, and glucuronides are eluted with ethyl ether containing 2% acetic acid. Glucuronide eluates are washed with 1% aqueous acetic acid to remove cross-reacting steroid sulfates. Assays of sera for the four steroids were performed using standard radioimmunoassay methodology, except for androsterone glucuronide. This steroid was assayed with a novel radioimmunoassay method that employees a tritiated, unconjugated androsterone tracer and an anti-dehydroepiandrosterone sulfate antiserum. The new method is well suited for the assay of conjugated and unconjugated steroids in large numbers of specimens, particularly where the sample volume is limited.