Detection of hormones (E1, E2, EE2) according to the requirements of the EU Water Framework Directive using an online-SPE-HPLC-MS/MS

Importance of the Topic

The presence of estrogenic hormones such as estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) in surface waters poses significant ecological and human health risks even at ultra-trace concentrations. Compliance with the EU Water Framework Directive requires detection limits of 400 pg/L for E1 and E2 and 35 pg/L for EE2. Achieving these thresholds in complex environmental matrices demands highly selective, sensitive, and automated analytical workflows.

Objectives and Study Overview

This study aimed to develop and validate an online solid-phase extraction (SPE) coupled with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method capable of reliably quantifying E1, E2 and EE2 at levels meeting or exceeding EU regulatory requirements. The approach was benchmarked against an alternative GC-MS/MS method.

Methodology

Sample Collection and Preparation

• One-liter surface water samples spiked with isotopically labelled internal standards (1 ng/L).
• Offline SPE enrichment using Speedisk C-18 cartridges followed by silica clean-up and solvent evaporation.
• Reconstitution in water and direct injection of 1 mL into the online SPE-HPLC-MS/MS system.

Chromatographic and MS Conditions

• Online SPE on a cyano-phase cartridge with gradient elution into an HPLC column (Raptor Biphenyl, 2.7 µm, 50×2.1 mm) at 0.4 mL/min.
• Mobile phase: acetonitrile/water with 0.05 mM ammonium fluoride.
• Negative electrospray ionisation MS/MS with multiple reaction monitoring (MRM) transitions selected for each analyte.

Instrumentation

The primary system comprised a Shimadzu LC-MS 8060 coupled with a Chronect µSPE module. As an alternative, Shimadzu GC-MS/MS TQ 8040 using derivatisation (BSTFA/pyridine) and MRM analysis on a Restek 5Sil-MS column was evaluated.

Main Results and Discussion

In reagent standards, theoretical limits of detection were calculated as 0.7 pg/L for EE2, 0.4 pg/L for E2 and 0.3 pg/L for E1. In real surface water matrices, practical LODs of 30 pg/L for EE2 and E2 and 10 pg/L for E1 were achieved. Spike recovery experiments at 30 pg/L EE2 confirmed these values. The method fulfilled EU requirements for all three hormones. GC-MS/MS also demonstrated sufficient sensitivity for regulatory monitoring.

Benefits and Practical Applications

The developed online SPE-HPLC-MS/MS workflow offers

• Automated sample clean-up and enrichment reducing manual steps and variability.
• High selectivity for matrix removal via cyano SPE and biphenyl chromatography.
• Low detection limits enabling compliance with stringent regulatory standards.
• Applicability for routine monitoring of endocrine disruptors in water bodies.

Future Trends and Potential Uses

Future developments may include

• Expansion to multiplexed analysis of a broader range of endocrine disruptors and emerging contaminants.
• Integration with high-resolution mass spectrometry for non-target screening and suspect analysis.
• Deployment of real-time online monitoring systems for early warning of water quality issues.
• Application of the method to diverse environmental matrices to further assess robustness.

Conclusion

The proposed online-SPE-HPLC-MS/MS method reliably quantifies E1, E2 and EE2 at ultra-trace levels, fulfilling EU Water Framework Directive requirements. The approach delivers high sensitivity, selectivity and automation, making it suitable for routine environmental monitoring. GC-MS/MS serves as a viable complementary technique.

References

1. EU 2018/840. Commission Implementing Decision (EU) 2018/840 of 5 June 2018 establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council and repealing Commission Implementing Decision (EU) 2015/495.
2. Loos R., Marinov D., Sanseverino I., Napierska D., Lettieri T. Review of the 1st Watch List under the Water Framework Directive and recommendations for the 2nd Watch List. EUR 29173 EN. Publications Office of the European Union, Luxembourg, 2018. ISBN 978-92-79-81839-4. doi:10.2760/614367.
3. Itzel F., Gehrmann L., Teutenberg T., Schmidt T.C., Tuerk J. Recent developments and concepts of effect-based methods for the detection of endocrine activity and the importance of antagonistic effects. Trends in Analytical Chemistry, 2019, 118:699–708.