Sexual Hormones Determination in Biofluids by In-Vial Polycaprolactone Thin-Film Microextraction Coupled with HPLC-MS/MS

TL;DR

A new method using a polycaprolactone film efficiently extracts sex hormones from biological samples like urine and serum for analysis.

Contribution

The study demonstrates the first application of in-vial polycaprolactone thin-film microextraction for sex hormones in biofluids using HPLC-MS/MS.

Findings

The polycaprolactone film achieved high recovery rates of unconjugated sex hormones in urine and serum within short extraction times.

The method requires dilution and washing steps for protein matrices to ensure clean extracts suitable for analysis.

The film is reusable up to ten cycles and aligns with green chemistry principles for sample preparation.

Abstract

The in-vial microextraction technique is emerging as an alternative sample treatment, as it integrates sorbent preparation, adsorption, and desorption of analytes in a single device before instrumental analysis. In this work, the applicability of polycaprolactone polymeric film, recently used for the in-vial microextraction of sex hormones from environmental waters, is studied in a low-capacity format for unconjugated sex hormones determination in biological samples by HPLC-MS/MS. Its performance was evaluated in urine and serum, achieving extraction in a short time (10 and 30 min, in turn) and satisfactory elution with ethanol, with recovery in the range of 65–111% in urine, 55–122% in bovine serum albumin (BSA) solution, and 66–121% in fetal bovine serum (FBS). In the case of protein matrices, a dilution to 20 g L−1 protein content and washing step (3 × 1 mL ultrapure water) afore the elution are required to achieve clean extract, as verified by a Bradford assay. Matrix-matched calibration was used for quantification, obtaining correlation coefficients greater than 0.9929; limits of detection and quantification were in the range of 0.01–0.65 and 0.03–1.96 ng mL−1 in urine, 0.02–0.8 and 0.05–2.5 ng mL−1 in BSA, and 0.02–1.0 and 0.06–3.0 g mL−1 in FBS, respectively. The in-vial polycaprolactone film proved to be reusable for several cycles (up to ten), and the greenness assessment revealed a good adhesion to green sample preparation principles. All these achievements further strengthen its feasibility for efficient extraction/clean-up of trace sex hormones in complex biological samples.

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