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

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.