Quantitative Analysis of Natural and Synthetic Estrogens in Surface and Final Effluent Waters at Low ppq Levels Using UPLC-MS/MS

Significance of the Topic

Estrogens, widely used in contraceptive therapies and hormone replacement, can enter aquatic systems through treated wastewater discharges. These compounds disrupt endocrine systems in fish and pose ecological risks. The EU has placed 17α-ethinylestradiol and 17β-estradiol on a watch list to better understand their occurrence and potential impacts at ultra-trace levels.

Objectives and Study Overview

This study aimed to develop and validate an analytical method for quantifying estrone, 17α-ethinylestradiol (EE2), and 17β-estradiol (E2) in surface and final effluent waters at parts-per-quadrillion levels, meeting EU-defined lower limits of quantitation (LLOQs). The approach combined sample enrichment, high-resolution separation, and sensitive mass spectrometric detection.

Methodology and Instrumentation

Sample preparation involved off-line solid phase extraction (SPE) using Oasis and Sep-Pak cartridges for cleanup and pre-concentration. After dry-down and reconstitution in LCMS-grade water, a 100 μl aliquot was injected onto an ACQUITY UPLC H-Class system with an extension loop:

• Column: ACQUITY UPLC BEH C18, 1.7 μm, 3.0×100 mm at 30 °C
• Mobile phases: water with 0.01 mM NH4F (A) and 50:50 acetonitrile:methanol with 0.01 mM NH4F (B)
• Gradient: 70% A (0–1 min), linear to 5% A (3.5 min), hold (5.5 min), re-equilibrate (8.6 min)
• Flow rate: 0.6 mL/min; sample temperature: 10 °C

Detection was performed on a Xevo TQ-XS triple quadrupole mass spectrometer in ESI-MRM mode. The integrated fluidics system diverted non-relevant flow to waste, protecting the source. Data acquisition and processing used MassLynx v4.2.

Main Results and Discussion

Optimized chromatographic conditions yielded baseline separation of the three estrogens in a 50 ng/L standard. Method calibration in surface water and final effluent demonstrated excellent linearity (R2>0.997) over 10–320 ng/L for EE2 and 62.5–2000 ng/L for E2 and estrone. Repeatability (%RSD) was below 6% for both matrices. SPE recoveries exceeded 80% with acceptable run-to-run variation. Matrix suppression ranged from −22% (surface water) to −72% (final effluent), but low-level analyte detection remained robust. The method achieved EU LLOQs in surface water at low pg/L levels. In final effluent, EE2 was quantified at 16.9 pg/L using a standard addition approach.

Benefits and Practical Applications

• High analytical sensitivity enabling compliance with stringent EU LLOQs
• Baseline separation and robust peak shapes for target analytes
• Reliable linearity and repeatability in complex environmental matrices
• Effective SPE recoveries and matrix cleanup
• Quantitation in absence of isotopically labeled standards via standard addition

These attributes support routine monitoring of estrogens in water quality and regulatory programs.

Future Trends and Potential Applications

Advancements may include incorporation of isotope-dilution techniques to further improve quantitation accuracy, automation and miniaturization of SPE workflows, and integration with high-resolution mass spectrometry for broader screening of endocrine-disrupting compounds. Data generated will inform ecological risk assessments and drive regulatory decisions.

Conclusion

This work establishes a robust UPLC-MS/MS method for quantifying estrone, 17α-ethinylestradiol, and 17β-estradiol at ultra-trace levels in surface and treated effluent waters. The combination of off-line SPE, large volume injection, and Xevo TQ-XS detection meets challenging EU LLOQs, offering a reliable tool for environmental monitoring and compliance.

References

1. Schwindt A, Winkelman D, Keteles L, et al. An Environmental Oestrogen Disrupts Fish Population Dynamics through Direct and Transgenerational Effects on Survival and Fecundity. Journal of Applied Ecology. 2014.
2. Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013 Amending Directive 2000/60/EC and 2008/105/EC in Respect of Priority Substances in the Field of Water Policy. August 2013.
3. Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 Establishing a Framework for Community Action in the Field of Water Policy. October 2000.
4. Commission Implementing Decision (EU) 2015/495 of 20 March 2015 Establishing a Watch List of Substances for Union-Wide Monitoring in the Field of Water Policy. March 2015.
5. Ross E, Boag A, Todd H, Gatward N. Analysis of Natural and Synthetic Estrogens at Sub-PPT Levels in Surface Water and Crude Influent Water Utilizing the ACQUITY UPLC System with 2D LC Technology and Xevo TQ-S. Waters Technology Brief No. 720005626en. March 2016.
6. Chang H, et al. Occurrence of Androgens and Progestogens in Wastewater Treatment Plants and Receiving River Waters: Comparison to Estrogens. Waters Research. 45(2):732–740. 2011.