Reversed-phase C18 cartridge for extraction of estrogens from urine and plasma

We describe a simple method for extraction of estrogens that is efficient over the whole polarity range of estrogen metabolites in urine and plasma. A small reversed-phase C18 cartridge (Sep-Pak C18, Waters Associates, Inc.) is used for this purpose. After application of the biological fluid, followed by an aqueous wash, estrogens are eluted in 3 mL of methanol. For urine the pH should be kept at 3 before the methanol elution. When estrogens are extracted from plasma, the steroid-protein interaction is counteracted by the presence of triethylammonium sulfate and D-norgestrel at pH 5. The recoveries of added 3H-labeled estrogens are essentially quantitative. The capacity of the column is high and estrogens can be extracted from large volumes of pregnancy urine without loss in the washing water. Because of the high flow rate (2 mL/min) and the small volume of methanol in which the estrogens are eluted, the whole procedure is convenient and rapid.