The Steroid Hormones Determination in Water

Importance of the topic

The presence of steroid hormones in water sources represents a growing concern for public health and environmental integrity. Even at trace levels, these endocrine-disrupting micropollutants can interfere with aquatic organisms’ reproduction and hormone regulation, and ultimately enter drinking water supplies.

Objectives and overview of study

This report presents a validated analytical workflow to detect and quantify five key steroid hormones—17β-estradiol (E2), 17α-estradiol, 17α-ethinylestradiol (EE2), equilin and estrone (E1)—in various water matrices. The aim is to meet stringent regulatory limits (1 ng/L) by combining efficient sample preparation and highly sensitive detection.

Methodology

Water samples (minimum 250 mL, collected in amber glass bottles) undergo off-line solid phase extraction (SPE) to achieve up to 1,000-fold concentration. The extracted analytes are eluted with organic solvent prior to instrumental analysis. Quantification is performed by ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) operating in electrospray ionization mode, employing multiple reaction monitoring for selectivity and sensitivity.

Used Instrumentation

• SPE manifold with cartridges tailored for steroid chemistry.
• UHPLC system with sub-2 µm particle column for rapid separation.
• Triple-quadrupole mass spectrometer with electrospray ion source.
• Automated autosampler and data acquisition software.

Main results and discussion

This method achieves detection limits down to 0.8–1 ng/L for all target hormones, meeting WHO and EU Directive 2020/2184 benchmarks. Validation demonstrated high recovery (75–95 %), precision (<15 % RSD) and minimal matrix effects in tap, bottled and surface water. Routine monitoring revealed occasional detections of 17β-estradiol and EE2 in treated wastewater effluents, underscoring the need for continued surveillance.

Benefits and practical applications

• Enables compliance testing against regulatory thresholds for endocrine disruptors.
• Applicable to drinking water, environmental monitoring and wastewater treatment efficacy studies.
• Offers a robust platform for routine QA/QC in water analysis laboratories.

Future trends and potential uses

Advances in on-line SPE-LC-MS/MS automation and high-resolution mass spectrometry promise further sensitivity gains and workflow streamlining. Emerging interest in non-targeted screening may expand detection to novel hormone derivatives and metabolites. Coupling bioassays with chemical analysis could provide comprehensive assessments of endocrine activity in water.

Conclusion

The described SPE-UHPLC-MS/MS method offers a reliable and sensitive approach for quantifying trace steroid hormones in diverse water matrices, supporting regulatory compliance and safeguarding public health. Continued methodological innovations will strengthen environmental monitoring and risk assessment of endocrine disruptors.

References

• United States Environmental Protection Agency. Method 539: Determination of hormones in drinking water by solid phase extraction and liquid chromatography–electrospray ionization tandem mass spectrometry.
• Goeury K, et al. Assessment of automated off-line solid phase extraction LC-MS/MS to monitor EPA priority endocrine disruptors in tap water, surface water, and wastewater. Talanta. 2022; DOI:10.1016/j.talanta.2022.123216.
• Molnár S, Kulcsár G, Perjési P. Determination of steroid hormones in water samples by liquid chromatography electrospray ionization mass spectrometry using parallel reaction monitoring. Microchemical Journal. 2022; DOI:10.1016/j.microc.2021.107105.