Combination of automatic HPLC‐RIA method for determination of estrone and estradiol in serum

Abstract

We developed a highly sensitive assay for estrone and 17 beta-estradiol in serum. Estrone and 17 beta-estradiol, obtained by solid-phase extraction using a Sep pak tC18 cartridge, were purified by high-performance liquid chromatography (HPLC). Quantitation of estrone and 17 beta-estradiol were carried out by radioimmunoassay. Not insignificantly, this automatic system of extraction and HPLC succeeded in analyzing 80 samples a week. Intra-assay coefficients of variation (CV) for estrone and 17 beta-estradiol ranged from 19.5 to 28.7%, and from 8.5 to 13.7%, respectively. The minimum detectable dose for estrone and 17 beta-estradiol were 1.04 pg/ml and 0.64 pg/ml, respectively. The serum levels of 17 beta-estradiol using our method strongly correlated with those by Gas chromatography mass spectrometry (GC-MS). The serum levels of estrone and 17 beta-estradiol in 154 peri- and postmenopausal women were estimated to be between 15 and 27 pg/ml and between 3.5 and 24.0 pg/ml, respectively, while the serum level of 17 beta-estradiol in postmenopausal women, in particular, was estimated to be from 3.5 to 6.3 pg/ml. For postmenopausal women who suffered from vasomotor symptoms, the mean levels of estrone and 17 beta-estradiol at 12 to 18 hours after treatment with daily 0.625 mg conjugated equine estrogen (CEE) and 2.5 mg medroxyprogesterone acetate (MPA) were 135.0 and 21.3 pg/ml at 12 months, respectively. On the other hand, levels of estrone and 17 beta-estradiol at 12 to 18 hours after treatment with CEE and MPA every other day, were 73.4 and 15.3 pg/ml, respectively. These highly sensitive assays for estrone and 17 beta-estradiol are useful in measuring low levels of estrogen in postmenopausal women, and monitoring estrogen levels in women receiving CEE as hormone replacement therapy.

Plasma oestrogen changes in adult male cats after orchiectomy, body-weight gain and low-dosage oestradiol administration

“…Plasma E 1 concentrations before OX were not significantly different after OX or E 2 administration (Table 1). These concentrations were approximately five- to ten-fold greater than E 2 concentrations, a condition observed in other species ( 8 ) .…”

“…Plasma E 1 and E 2 concentrations were determined by a method developed for use on human plasma ( 8 ) with the following modifications: plasma samples (1 ml) were solid-phase extracted, dried at 35°C by centrifugal evaporation, reconstituted in 2-propanol and injected (40 μl) on an HPLC column (Capcell Pak NH2 UG80 S5; Shiseido, Tokyo, Japan). E 1 and E 2 fractions were isocratically eluted at 1 ml/min with a mobile phase, 2-propanol–hexane (3:7, v/v, HPLC grade; Fisher Scientific, Fair Lawn, NJ, USA), for which the residue left after drying did not interfere with RIA of the oestrogens.…”

Estradiol levels in prepubertal boys and girls – analytical challenges

“…Another approach to increase assay sensitivity is the combination of high-performance liquid chromatography (HPLC) and RIA. The purifying HPLC step enables the usage of an antiserum with a high affinity rather than a high specificity in the following RIA step (Yasui et al, 1999).…”

Droplet-Scale Estrogen Assays in Breast Tissue, Blood, and Serum

“…This sample size is 1000 to 4000 times smaller than that required for conventional methods of extrac- tion and quantification of steroids, including extraction followed by immunoassays or MS (8,9,(34)(35)(36)(37) (fig. S1).…”