This study examined the effects of ionic strengths of NaCl (0.1, 0.3, and 1.0？M), pH (3, 7, and 11), and organic solvents (dichloromethane, diethyl ether, and methanol) on the extraction of estradiol at concentrations of 5.0？pg/mL in human serum. Methanol extracted almost 100% of the estradiol at a 5.0？pg/mL concentration, while ether and dichloromethane extracted only 73% or 70%, respectively, of the estradiol. The methanol extracted material was subjected to reverse phase high-performance liquid chromatography (HPLC) using 60% methanol and was found to elute at the same position as estradiol standard. These results suggest that methanol extraction of estradiol may prove useful in situations where estradiol occurs at concentration levels of ≥5.0？pg/mL, concentrations of great clinical significance in the detection and treatment of breast cancer. 1. Introduction The routine measurement of serum estradiol levels in postmenopausal women requires an assay with a sensitivity of less than 5.0？pg/mL because postmenopausal women produce estradiol but not from their ovaries and therefore have extremely low serum levels [1]. There are practical analytical limitations to the estradiol assay when applied to clinical settings where levels may be ≤5.0？pg/mL. Present methods of estradiol analysis offer sensitivity of >20.0？pg/mL, which clearly points to the need for better routine methods of estradiol extraction and analysis at concentrations of 5.0？pg/mL or less [2]. The present study examined the effect of ionic strengths of NaCl (0.1？M, 0.3？M, and 1.0？M), pH (3, 7, and 11), and organic solvents (dichloromethane, diethyl ether, and methanol) on the extraction and analysis of estradiol at concentrations of 5.0？pg/mL. 2. Materials and Methods 2.1. Materials All chemical reagents and solvents were obtained from Fisher Scientific Co. (Valley Stream Parkway, Malvern, PA, USA). 125I labeled estradiol was obtained from Diagnostic Systems Laboratories, Inc. (Webster, TX, USA). 2.2. Methods 2.2.1. Preparation of Stripped Serum The whole and “stripped” human serum was prepared at Massachusetts General Hospital, Boston, MA, USA. Briefly, 100？mL samples of freshly prepared serum were mixed with 30 grams of activated charcoal (G-60) and 6.0 grams of dextran (Pharmacia) for 30 minutes at 4°C. The mixture was centrifuged at 20,000？×g for 30 minutes at 4°C. The supernatant was passed through a 0.45 micron filter and stored frozen until use. The concentration of estradiol in this original stock prior to dilution was determined as described elsewhere [3, 4]. 2.2.2. Extraction
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