# Development of an HPLC-FLD Method for Estradiol and Metabolites: Application of Solid-Phase Microextraction

## TL;DR

Researchers developed a sensitive HPLC method with fluorescence detection to measure estradiol and its metabolites in biological samples using solid-phase microextraction.

## Contribution

A novel HPLC-FLD method with SPME for estradiol and metabolite quantification in serum and saliva is introduced.

## Key findings

- The method achieved good linearity for estradiol and its metabolites with R² values between 0.9893 and 0.9995.
- The limit of quantification was 10 ng/mL for all analytes.
- Solid-phase microextraction outperformed liquid–liquid extraction in this application.

## Abstract

Estrogens are potent hormones involved in numerous physiological and pathological processes. Their typically low concentrations in biological samples necessitate highly sensitive analytical methods for accurate quantification. This study presents a high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for quantifying estradiol and its metabolites in blood serum and saliva. Analytes were extracted using solid-phase microextraction with a divinylbenzene sorbent and methanol as the desorption agent. FLD was performed after the derivatization of the analytes with dansyl chloride. Separation was achieved on a Poroshell 120 EC-C18 column (2.1 × 100 mm, 2.7 µm) at 50 °C using water with 0.1% formic acid and methanol as the mobile phase at 0.5 mL/min. A gradient elution increased the methanol concentration from 76% to 100% over 0–8 min, then it returned to 76% at 8.1 min and was held until 11 min had passed. Detection was at λEX 350 nm and λEM 530 nm. Good linearity was observed for estradiol, 2-hydroxyestradiol, and 2-methoxyestradiol (10–300 ng/mL; R2 = 0.9893–0.9995). The LOQ for all analytes was 10 ng/mL. Solid-phase microextraction (SPME) offered advantages over liquid–liquid extraction. The method is suitable for quantifying estrogens in the 10 ng/mL–1 µg/mL range.