Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Jul;48(7):e70229.
doi: 10.1002/jssc.70229.

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

Lucas Morés  1   2   3 , Camila Will  3 , Eduardo Carasek  3 , Josias Merib  1   2
Affiliations

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

Lucas Morés et al. J Sep Sci. 2025 Jul.

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.

Keywords: bioanalytical methods; estrogens; microextraction; sustainability; urine monitoring.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflicts of interest.

Figures

FIGURE 1
FIGURE 1
NADES evaluated for the extraction of steroidal hormones from urine samples, as follows: (2) Thy:C10; (3) Ca:C10; (4) L:Thy; (6) C12:C6; (8) Thy:Cam. Experimental conditions: urine adjusted to pH 10; extraction time of 60 min; desorption time of 20 min; acetonitrile as desorption solvent; analytes spiked at 500 ng mL−1.
FIGURE 2
FIGURE 2
Response surface obtained for the optimization of desorption solvent. Experimental conditions: urine adjusted to pH 11; extraction time of 60 min; desorption time of 20 min; analytes spiked at 500 ng mL−1; NADES—Thy:Cam (1:1 v/v).
FIGURE 3
FIGURE 3
Response surface obtained for the optimization of extraction time and sample pH. Experimental conditions: desorption time of 30 min; acetonitrile as desorption solvent; analytes spiked at 500 ng mL−1; NADES—Thyl:Cam (1:1 v/v).
FIGURE 4
FIGURE 4
Evaluation of the sustainable aspects of the methodology using (A) ComplexGAPI and (B) AGREEprep.
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Suidan M. T., Esperanza M., Zein M., McCauley P., Brenner R. C., and Venosa A. D., “Challenges in Biodegradation of Trace Organic Contaminants—Gasoline Oxygenates and Sex Hormones,” Water Environment Research 77 (2005): 4–11, 10.2175/106143005X41564. - DOI - PubMed
    1. Tomšíková H., Aufartová J., Solich P., Sosa‐Ferreira Z., Santana‐Rodríguez J. J., and Nováková L., “High‐Sensitivity Analysis of Female‐Steroid Hormones in Environmental Samples,” TrAC Trends in Analytical Chemistry 34 (2012): 35–58, 10.1016/j.trac.2011.11.008. - DOI
    1. Viglino L., Aboulfadl K., Prévost M., and Sauvé S., “Analysis of Natural and Synthetic Estrogenic Endocrine Disruptors in Environmental Waters Using Online Preconcentration Coupled With LC‐APPI‐MS/MS,” Talanta 76 (2008): 1088–1096, 10.1016/j.talanta.2008.05.008. - DOI - PubMed
    1. Denver N., Khan S., Homer N. Z. M., MacLean M. R., and Andrew R., “Current Strategies for Quantification of Estrogens in Clinical Research,” Journal of Steroids Biochemistry and Molecular Biology 192 (2019): 105373, 10.1016/j.jsbmb.2019.04.022. - DOI - PMC - PubMed
    1. do Carmo S. N., Merib J., and Carasek E., “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,” Journal of Chromatography B 1118–1119 (2019): 17–24, 10.1016/j.jchromb.2019.04.037. - DOI - PubMed

LinkOut - more resources