Environmentally Friendly Hydrophobic Deep Eutectic-Based Solvent Bar Microextraction Combined with HPLC-DAD for Determination of Steroid Hormones in Urine and Water

Abstract

Steroid hormones may pose potential risks to both human health and wildlife, primarily through the consumption of medication or polluted food and water; efforts are being made to monitor their levels in the human body and regulate and minimize their releases to the environment. In this study, a simple and environmentally friendly sample preparation method was developed to simultaneously determine three steroid hormones in urine and water samples. A monoterpene (menthol) and a fatty acid (lauric acid) were combined in various ratios to form a hydrophobic-deep-eutectic (HDE) as an extraction solvent in solvent-bar-microextraction (SBME). Using a univariate strategy, the menthol-to-lauric acid HDE ratio of 4:1 and pH 7 of the sample solution resulted in the highest extraction efficiency (EE%) of selected steroids. Computational methods have been employed to predict 4:1 HDE interaction with chosen steroids. Additionally, chemometric approaches suggested that the optimal extraction conditions involved HDEs as extract solvent confined within 3 SBME devices directly immersed into 20 mL sample solution with 30 min extraction time followed by ultrasonication within 200 μL of elution solvent for 5 min elution time. Under optimized conditions, the method calibration graph for the spiked selected steroids in water and urine samples showed good linearity with R2≥ 0.994 with limits of detection/ quantification lower than 0.40/ 1.35 μg L-1 and Repeatability/ reproducibility (RSD%, n=5) lower than 5.09/ 7.11. The developed method allows safe, rapid, and reliable analysis of three steroid hormones in human urine and water samples without using toxic volatile organic solvents.

Keywords: Hydrophobic deep eutectic solvent, Solvent bar microextraction, Steroid hormones, HPLC-DAD, Urine and water