A Membrane-Based Strategy for the High-Throughput Determination of Steroidal Hormones in Human Urine Using Natural Deep Eutectic Solvents Combined With Liquid Chromatography Coupled With Diode Array Detector

Abstract

Estrogen hormones are present in the human body at varying concentrations, either naturally or through ingestion. At regulated levels, these compounds perform vital functions in the body. However, elevated concentrations are related to deregulation of several processes, such as abnormal cell proliferation, which can lead to the development of cancer (breast and ovarian) and other diseases (endometriosis). In this work, an analytical methodology is proposed for the determination of 17β-estradiol (E1), estrone (E2), and 17α-ethinylestradiol (EE2) in human urine. Hollow fiber-microporous membrane liquid–liquid extraction (HF-MMLLE) is used with natural deep eutectic solvents (NADESs). This technique was associated with a 96-well plate system followed by high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD). The optimized conditions consisted of using thymol and camphor (1:1 v/v) as extraction solvent; an extraction time of 100 min; acetonitrile as desorption solvent; a desorption time of 30 min; and a sample pH adjusted to 12. Limits of quantification (LOQs) of 25 ng mL⁻¹ for E2 and 50 ng mL⁻¹ for E1 and EE2, and limits of detection (LOD) of 7.5 ng mL⁻¹ for E2 and 15 ng mL⁻¹ for E1 and EE2 were obtained. Intraday and interday precisions were lower than 8.7% and 21.6%, respectively. Relative recoveries were examined in two samples and ranged from 68.2% to 131.8%. The method was applied in samples from healthy volunteers, and concentrations from 75.6 to 182.7 ng mL⁻¹ for E2 were found. Finally, the method was evaluated by two sustainable metrics to examine the green aspects of the experimental approach.