AbstractA procedure is described for the analysis of the estrogens equilin and estrone in human plasma following oral administration of conjugated estrogen preparations. After enzymatic hydrolysis of the sulfate conjugates, plasma proteins are precipitated with methanol and the estrogens extracted into ethyl acetate. Derivatization with the reagent flophemesylamine converts equilin and estrone into volatile pentafluorophenyldimethylsilyl ethers ideally suited to capillary gas chromatography‐negative ion chemical ionization mass spectrometry. Using a 15 meter dimethyl silicone bonded phase fused silica capillary column separation of the estrone and equilin derivatives is achieved within 9 minutes. Selected ion monitoring of the intense negative molecular ions enables levels of 1 ng.ml−1to be measured with coefficients of variation of 9.3 % and 14.2 % for estrone and equilin respectively. Plasma levels of the compounds are reported in two male volunteers up to 24 hours after dosing with 5 milligrams of Premarin™. (™ Ayerst Laboratories Inc., New York