Quantification of hormones (E1, E2, EE2) according to the requirements of the EU Water Framework Directive using on-line-SPE-HPLC-MS/MS [LCMS-8060NX]

Significance of the Topic

The presence of estrogenic hormones such as estrone (E1), 17-β-estradiol (E2) and 17α-ethinylestradiol (EE2) in surface waters poses ecological and human health risks. These substances are included on the EU Water Framework Directive Watch List, which mandates sensitive monitoring at ultra-trace levels. Developing robust analytical workflows to reliably quantify these hormones in complex aqueous matrices is critical to meet regulatory limits and support environmental quality assessments.

Objectives and Study Overview

This study aimed to establish and validate a highly selective on-line solid phase extraction (SPE) coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantifying E1, E2 and EE2 in river water influenced by wastewater effluent. Target detection limits of 0.4 ng/L for E1 and E2, and 0.035 ng/L for EE2 were defined by the EU Directive. Method performance was demonstrated by spiking samples at 0.030 ng/L (below the EE2 limit) and 0.090 ng/L (three-times the EE2 limit) to evaluate sensitivity, recovery and repeatability.

Methodology and Instrumentation

Sample Collection and Preparation:

• One-liter surface water samples were collected from a river with significant wastewater input.
• Isotopically labeled internal standards were added before any cleanup.
• Off-line enrichment was conducted by C18 solid phase extraction (Speedisk C-18 cartridges) and eluted with n-hexane/ethyl acetate.
• A silica cartridge cleanup step removed residual lipophilic interferences.
• Extracts were evaporated and reconstituted in 2 mL of aqueous phase.

On-line SPE and LC-MS/MS Analysis:

• An on-line SPE unit was integrated with a Nexera X3 UHPLC for further cleanup and concentration.
• Chromatographic separation employed 0.05 mM ammonium fluoride and acetonitrile gradients.
• Detection was performed on a Shimadzu LCMS-8060NX in negative electrospray ionization mode with 1.5 mL injection volume.
• Multiple reaction monitoring (MRM) transitions were optimized for each analyte to achieve high selectivity.

Main Results and Discussion

Calibration and Linearity:

• Linear ranges covered 0.005–45 ng/L for EE2, 0.009–9 ng/L for E2 and 0.009–1.8 ng/L for E1, with correlation coefficients (r²) ≥ 0.9992.
• Calibration curves showed consistent slopes and low relative standard deviations (%RSD < 6 % across replicates).

Sensitivity and Repeatability:

• Signal-to-noise ratios at 0.030 ng/L were > 8 for EE2 and detectable for E1/E2 despite matrix noise.
• Repeatability over 12 injections yielded %RSDs of 12–20 % for peak area ratios.

Recovery and Matrix Effects:

• Recoveries ranged from 73 % to 126 % for all analytes at both spike levels, demonstrating reliable quantification in surface water.
• Chromatographic resolution fully separated isobaric E2 and 17α-estradiol peaks, avoiding misidentification.

Benefits and Practical Applications of the Method

This on-line SPE-LC-MS/MS approach meets and surpasses the EU Watch List detection requirements while offering:

• High sensitivity for ultra-trace hormone quantification.
• Enhanced selectivity through tandem cleanup and MRM transitions.
• Sufficient robustness for routine laboratory analysis of complex water samples.

Future Trends and Applications

Emerging directions include:

• Expansion to other endocrine disrupting compounds and polar contaminants.
• Automation of sample cleanup and injection to increase throughput.
• Integration with high-resolution mass spectrometry for non-target screening.
• Alternative GC-MS/MS workflows for volatile or derivatizable analytes.

Conclusion

The developed on-line SPE-HPLC-MS/MS method reliably quantifies trace levels of E1, E2 and EE2 in surface water according to EU Water Framework Directive requirements. Its high sensitivity, precision and robustness make it well suited for routine environmental monitoring.

References

1. EU 2018/840, Official Journal of the European Union, 2018, L141/9-12
2. Itzel, F. et al., Trends in Analytical Chemistry, 2019, 118:699-708