Immunoaffinity extraction for liquid chromatographic determination of equilin and its metabolites in plasma

Immunoaffinity extraction for the high-performance liquid chromatographic determination of equilin and its metabolites in plasma has been achieved. The antibody raised against an equilin 3-O-carboxymethyl ether-bovine serum albumin conjugate was characterized as having a high affinity for equilin and equilenin. One mL of the immunoaffinity adsorbent prepared by immobilization of an antibody was capable of retaining up to 1 microgram of equilin and equilenin, to 100 ng of other metabolites including 2-methoxylated and 17 beta-reduced compounds, and to 0.3 micrograms of glucuronic acid conjugates at C-3. The adsorbates were recovered qualitatively by elution with 90% aqueous (v/v) methanol without any interfering peaks on the chromatogram.