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
do Carmo, Sangela Nascimento; Merib, Josias; Carasek, Eduardo ✉
In this study, an environmentally friendly and high-throughput method was developed
for the determination of estrone (E1), 17 beta-estradiol (E2), 17 alpha-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/ 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 mu g L-1 for estradiol
to 3 mu g L-1 for estrone, while limits of quantification varied from 1 mu g L-1 for
estradiol to 10 mu g L-1 for estrone. The correlation coefficients ranged from 0.9947
for estrone to 0.9999 for estriol. The accuracy and intraassay and intermediate precisions
(RSD) were evaluated through extractions in diluted urine samples (40-fold) spiked
with each analyte (1, 200 and 400 mu g L-1 for E3; 0.1, 200 and 400 mu g L-1 for E2;
0.5, 200 and 400 mu g L-1 for EE2 and 10, 200 and 400 mu g L-1 for El). 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.