Method development, validation, and impurity measurement of ß-estradiol from radiolabeled [131I]ß-estradiol using radio-high-performance liquid chromatography for radioligand of saturation binding assay.

ß-estradiol is an estrogen steroid hormone and acts as an estrogen receptor agonist. Radiolabeled ß-estradiol is widely used as a radioligand for binding assays. In this present study, the synthesis of [131I]ß-estradiol has been successfully carried out. Accordingly, the measurement of the radiochemical purity (RCP) value and the presence of chemical impurities are needed. To validate the method for identifying the RCP and chemical impurities from [131I]ß-estradiol using high-performance liquid chromatography (HPLC). The synthesis of [131I]ß-estradiol was accomplished by a radioiodination reaction, and the RCP was determined by radio-HPLC. The method for ß-estradiol measurement was validated by reversed-phase HPLC radio-analytical employing ultraviolet-visible (UV-Vis) and radioactive detector. The method for radio-HPLC analysis was validated and established using a C-18 column and MeCN: H2O (55:45 v/v) as the mobile phase. The following conditions were applied: a flow rate of 1.2 mL/min, isocratic, and a UV-Vis detector at 280 nm. The RCP of [131I]ß-estradiol measured by thin-layer chromatography and radio-HPLC was 99.27% ± 1.25% and 95.75% ± 2.41%, respectively. The validation parameters were appropriate and met the requirements for acceptance. HPLC analysis was able to identify the presence of unlabeled estradiol (24.51%-27.29%) in the mixture of [131I]ß-estradiol. As a result, purification using preparative HPLC or other methods will be required in future studies.

Synthesis, stability, and cellular uptake of 131I-estradiol against MCF7 and T-47D human cell lines as a radioligand for binding assay.

Estradiol is a steroid hormone that works as an agonist estrogen receptor (ER). This compound is widely used as a ligand and bind specifically to the ERα. Radioligand binding assay is an in vitro method for drug development from natural products by synthesizing estradiol through radiolabeling using the radioiodination method. Synthesis of 131I-estradiol was perfomed by direct method using chloramine T as an oxidizer and by indirect labeling using 131I-histamine. The purity of chemical was determined by thin-layer chromatography and paper electrophoresis, as well as its stability for 30 days of storage in refrigerator, freezer and room temperature. The cellular uptake test of the radioligands from both methods was carried out with MCF7 and T-47D cell lines at 60 min. The results exhibited that 131I-estradiol was succesfully obtained with radiochemical purity greater than 95% and more stable in the refrigerator until 21 days than freezer and room temperature. 131I-estradiol and 131I-his-estradiol were internalized higher in T-47D cells than MCF7 cells (44.34 ± 5.93% vs. 17.27 ± 1.71% and 45.34 ± 6.42% vs. 4.92 ± 1.59%, respectively). Furthermore, the radioligands can be used to binding assay in determining the agonist or antagonist to ER of new drugs development.