Short communicationDetermination of urinary 15α-hydroxyestrogen levels via immunoaffinity extraction

- A method for 15α-hydroxyestrogen (OHE) quantification in rat urine was developed.
- Immunoaffinity column extraction and HPLC-electrochemical detection were used.
- We quantified three types of 15α-hydroxyestrogens with 97-109% precision.
- We believe this method can be used to better monitor fetal health.
- Our method could clarify inter-species and -sex differences in 15α-OHE levels.

15α-Hydroxyestrogens (15α-OHEs) are metabolites of the female hormone estradiol. In this study, to discover physiological markers that can be utilized for monitoring fetal conditions and estrogen-induced cancers, we established a method for quantifying 15α-OHEs in rat urine via immunoaffinity column extraction and HPLC-electrochemical detection, and detected 15α-OHEs in urine obtained male rats treated with estradiol. Notably, the standard curves for quantification obtained using the column were linear over a range of 0.5-50 ng 15α-OHEs. The accuracy of the analytical method with cleanup was 97-109% for the three kinds of 15α-OHEs examined, and the intra-assay precision of the measured values had a coefficient of variation of ≤20.6%. Therefore, the theoretical limit of quantification was 0.5 ng. However, the actual measured values obtained from the urine of male rats indicated that the detection limits were 0.425, 0.103, and 0.047 ng for estetrol, 15α-hydroxyestradiol, and 15α-hydroxyestrone, respectively. Our newly established method for measuring 15α-OHE concentrations in urine could facilitate characterization of the in vivo metabolic profile of 15α-OHEs in mammals under various physiological conditions, which could comprise clinical markers for monitoring human fetal health conditions in mammals.