Bract as a novel extraction phase in thin-film SPME combined with 96-well plate system for the high-throughput determination of estrogens in human urine by liquid chromatography coupled to fluorescence detection

In this study, an environmentally friendly and high-throughput method was developed for the determination of estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2) and estriol (E3) in human urine by liquid chromatography-fluorescence detector (HPLC-FLD). A biosorbent (bract) was proposed as extraction phase for Thin-Film SPME combined with 96-well system. The characterization of the biosorbent was performed by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The optimizations were carried out through univariate and multivariate approaches with optimal conditions comprised of urine samples diluted 40-fold, liquid desorption performed in methanol and addition of 20% (w/v) of NaCl in the sample. Considering an extraction/desorption cycle using the 96-well plate system, the sample preparation time was 1.7 min per sample, which contributes to the high-throughput of the method proposed. The analytical parameters of merit were determined and satisfactory results were achieved, including limits of detection ranging from 0.3 μg L^-1 for estradiol to 3 μg L^-1 for estrone, while limits of quantification varied from 1 μg L^-1 for estradiol to 10 μg L^-1 for estrone. The correlation coefficients ranged from 0.9947 for estrone to 0.9999 for estriol. The accuracy and intra-assay and intermediate precisions (RSD) were evaluated through extractions in diluted urine samples (40-fold) spiked with each analyte (1, 200 and 400 μg L^-1 for E3; 0.1, 200 and 400 μg L^-1 for E2; 0.5, 200 and 400 μg L^-1 for EE2 and 10, 200 and 400 μg L^-1 for E1). The relative recoveries (n = 3) ranged from 71 to 105%, intra-assay precision (n = 3) varied from 1 to 17% and intermediate precision (n = 9) ranged from 2 to 19%. The method developed can be successfully used for the quantification of estrogens in human urine samples.