Automated Extraction and Determination of Human Hormones in Drinking Water Using Solid-Phase Extraction and HPLC with UV Detection

Significance of the Topic

The occurrence of natural and synthetic human hormones in drinking water has raised considerable concern due to their potential to disrupt endocrine systems at low concentrations. Regulatory bodies such as the U.S. EPA and the European Parliament have highlighted several estrogens and androgens as priority contaminants. Reliable monitoring methods are needed to assess human exposure, understand long-term and synergistic effects, and develop effective treatment strategies.

Study Objectives and Overview

The primary goal of this study was to demonstrate the complete recovery of seven key human hormones—estriol, estrone, β-estradiol, 17α-ethynylestradiol, equilin, androstenedione, and testosterone—from fortified drinking water samples. The work employed automated solid-phase extraction (SPE) cartridges on a Dionex AutoTrace 280 system followed by HPLC with UV detection to evaluate the method’s performance in terms of sensitivity, precision, and accuracy.

Methodology and Instrumentation

Automated SPE was carried out using Thermo Scientific™ Dionex™ SolEx™ HRPHS polymeric cartridges on the Dionex AutoTrace 280 instrument. Each 20 mL water sample was loaded at 5 mL/min, rinsed, and eluted with 5 mL acetonitrile. Eluates were transferred to HPLC vials for analysis.

• SPE Instrument: Dionex AutoTrace 280 with a six-position cartridge manifold.
• Cartridges: SolEx HRPHS, 200 mg/6 mL, polymeric divinylbenzene-based with polyvinylpyrrolidone grafting.
• HPLC System: Thermo Scientific™ UltiMate™ 3000 RSLC—DGP-3600M pump, WPS-3000TRS sampler, TCC-3000RS column compartment, DAD-3000RS detector.
• Column: Acclaim™ 120 C18 guard (2.1 × 10 mm, 5 µm) and analytical (2.1 × 150 mm, 2.2 µm) columns.
• Mobile Phase: Water (A) and acetonitrile (B); gradient from 10% to 55% B (0–4 min), hold, then to 100% B (12–16 min), re-equilibration.
• Detection: UV at 214 nm; injection volume 2 µL; flow rate 0.2 mL/min; column temperature 20 °C.

Main Results and Discussion

The method achieved baseline resolution (Rs >1.5) for all hormone pairs on the C18 column at 214 nm. Linearity was excellent across 0.05–10 mg/L (r² >0.999). Limits of detection ranged from 0.025 to 0.40 mg/L, while quantification limits were 0.083–1.3 mg/L. Recoveries from fortified water averaged between 90% and 124% with RSDs below 5%, meeting EPA Method 539 criteria (70–130% recovery). Variability among cartridges was attributed to minor differences in eluent volume, yet overall performance remained robust. Increasing sample volumes to several liters could enhance sensitivity by up to three orders of magnitude for trace-level monitoring.

Benefits and Practical Applications

This automated SPE-HPLC-UV approach streamlines the preparation of aqueous samples containing low-level hormones. High recoveries, reproducible performance, and compatibility with EPA Method 539 make the workflow suitable for routine laboratory monitoring in environmental and drinking water quality control.

Future Trends and Potential Applications

Further integration with mass spectrometry detection could improve selectivity and lower quantification limits. Scaling up sample volumes and applying similar polymeric SPE phases to other emerging endocrine disruptors or pharmaceuticals can expand the utility of this platform. Coupling with online SPE and high-resolution MS may support comprehensive screening in environmental surveillance.

Conclusion

The combination of Dionex SolEx HRPHS cartridges on the AutoTrace 280 system and UltiMate 3000 RSLC with UV detection provides a robust, high-throughput method for extracting and quantifying human hormones in drinking water. The demonstrated sensitivity, accuracy, and automation support its application for regulatory compliance and environmental risk assessment.

Reference

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