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

Significance of the Topic

Trace levels of estrogenic compounds such as estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2) pose a significant environmental and public health concern due to their endocrine-disrupting potential. The EU Water Framework Directive has placed these substances on a watch list, mandating stringent detection limits to safeguard water quality and aquatic ecosystems. Developing robust analytical methods capable of reliably quantifying these hormones at ultra-trace concentrations is critical for regulatory compliance and monitoring efforts.

Objectives and Study Overview

The primary goal of this study was to establish an automated, online solid-phase extraction (SPE) coupled with high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method that meets or surpasses the EU-mandated limits of detection (400 pg/L for E1 and E2; 35 pg/L for EE2). The approach was validated using spiked surface water samples, focusing on improving selectivity, sensitivity and method robustness in complex matrices.

Methodology

Surface water (1 L) was collected and spiked with isotope-labelled internal standards. An initial offline enrichment employed C18 cartridges eluted with a 50:50 n-hexane/ethyl acetate mixture, followed by a silica cleanup step. The eluate was evaporated under nitrogen at 50 °C and reconstituted in water. The final extract underwent online SPE using a cyano (CN) cartridge, with analyte transfer to a biphenyl HPLC column under a gradient of acetonitrile and water (0.05 mM NH4F). Mass spectrometric detection utilized negative-mode electrospray ionization and multiple reaction monitoring (MRM) transitions specific to each estrogen.

Instrumentation

• Online SPE-HPLC-MS/MS system: Shimadzu LC-MS 8060 with Raptor Biphenyl column (50 × 2.1 mm, 2.7 µm) and CN SPE cartridge.
• Offline cleanup: Speedisk C18 and silica cartridges.
• Alternative confirmation: Shimadzu GC-MS/MS TQ 8040 with Restek 5Sil-MS capillary column and derivatization (BSTFA/pyridine).

Key Results and Discussion

Theoretical instrument detection limits derived from signal-to-noise of low-level standards were 0.3 pg/L for E1, 0.4 pg/L for E2 and 0.7 pg/L for EE2. In real surface water matrices, practical detection limits were determined at 10 pg/L for E1 and 30 pg/L for both E2 and EE2, validated by spiking experiments at 30 pg/L EE2. The online SPE-HPLC-MS/MS method successfully achieved the EU target sensitivity for all three analytes. A comparison with GC-MS/MS confirmed that both techniques offer viable routes to meet regulatory requirements, although LC-MS/MS provides a more streamlined workflow without derivatization.

Benefits and Practical Applications

This method delivers high selectivity through dual SPE cleanup and biphenyl chromatography, yielding improved signal-to-noise ratios in complex water samples. Automation of online SPE reduces hands-on time and potential errors, facilitating routine monitoring in environmental laboratories. The approach can be integrated into QA/QC protocols for compliance testing under the EU Water Framework Directive.

Future Trends and Potential Applications

Expanding this methodology to a broader range of estrogenic and micropollutant targets could streamline multi-residue monitoring programs. Incorporation of high-resolution mass spectrometry may further enhance identification of transformation products. Coupling with effect-based bioassays could provide comprehensive assessment of endocrine activity. Portable or miniaturized SPE-LC-MS systems may enable on-site screening in remote locations.

Conclusion

An online-SPE-HPLC-MS/MS workflow has been developed that meets the stringent EU Water Framework Directive requirements for ultra-trace quantification of E1, E2 and EE2 in surface waters. The method demonstrates robust performance in real matrices and offers an efficient, automatable solution for routine environmental monitoring. Broader validation across diverse water bodies and extension to additional analytes represent logical next steps.

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 and repealing Implementing Decision (EU) 2015/495.
2. Loos, R., Marinov, D., Sanseverino, I., Napierska, D., & Lettieri, T. (2018). 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.
3. Itzel, F., Gehrmann, L., Teutenberg, T., Schmidt, T. C., & Tuerk, J. (2019). Recent developments and concepts of effect-based methods for the detection of endocrine activity and the importance of antagonistic effects. Trends in Analytical Chemistry, 118, 699–708.