NEMC: Rapid Determination of Ethynylestradiol (17EE2) to 15 pg/L in wastewater using Thermo Scientific EQuan MAX Plus LC-MS on-line SPE and Thermo Scientific Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometer

Importance of the Topic

Endocrine-disrupting compounds such as 17α-ethynylestradiol (17α-EE2) pose significant risks to aquatic ecosystems and drinking water supplies due to their high estrogenic potency. Regulatory frameworks like the EU Water Framework Directive mandate detection limits as low as 35 pg/L, driving the need for highly sensitive, selective, and rapid analytical methods.

Objectives and Study Overview

This study aims to evaluate an on-line solid-phase extraction (SPE) method using the Thermo Scientific EQuan MAX Plus system coupled to a Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometer. Key goals include achieving or surpassing the EU Detection Limit for 17α-EE2, confirming its identification via MS2 fragments, assessing chromatographic resolution of isomers, and reducing total analysis time.

Methodology and Instrumentation

The workflow utilizes a 5 mL wastewater sample subjected to on-line SPE on the EQuan MAX Plus platform. Extracts are transferred directly into a Thermo Scientific UHPLC system equipped with:

• Loading column: Accucore aQ, 5 µm, 5 × 2.1 mm
• Analytical column: Acclaim VANQUISH Polar Advantage, 2.2 µm, 150 × 2.1 mm

Chromatography is performed at 0.3 mL/min with a gradient of 0.3 mM ammonium fluoride in water and methanol. Mass spectrometry employs negative heated electrospray ionization and Parallel Reaction Monitoring (PRM) targeting the precursor m/z 195.1705. MS/MS fragments (notably m/z 145.0660) are acquired at 70 000 resolving power, with mass accuracy maintained within 5 ppm.

Key Results and Discussion

The method achieves:

• Limit of Detection (LoD): 15 pg/L
• Limit of Quantitation (LoQ): 29 pg/L
• Calibration linearity: R² = 0.9998 across 25–1600 pg/L
• Mass accuracy: < 1 ppm error
• Precision: RSD 3.4% (n = 6 at 100 pg/L)
• Total analysis time: < 30 minutes (∼30× faster than conventional workflows)

Application to a real wastewater effluent sample detected 17α-EE2 at 462 pg/L. Preliminary data indicate potential chromatographic separation of 17α-EE2 and its 17β-isomer, which could prevent overestimation in monitoring programs.

Benefits and Practical Applications

This streamlined approach offers significant advantages:

• High sensitivity and selectivity for trace-level estrogens
• Reduced sample volume and reagent consumption
• Rapid throughput facilitating tighter sampling schedules
• Robust confirmation via high-resolution MS/MS
• Potential for isomer-specific quantitation

Future Trends and Applications

Further method enhancements may include incorporation of deuterated internal standards to improve quantitation robustness, expansion to other priority steroid estrogens (e.g., estrone, 17β-estradiol), and adaptation for automated, high-throughput environmental monitoring workflows. Advances in portable high-resolution MS could enable field-based screening of endocrine disruptors.

Conclusion

The combined on-line SPE UHPLC–Orbitrap MS method demonstrates superior sensitivity, accuracy, and speed for the determination of 17α-EE2 in wastewater, meeting stringent regulatory requirements and offering a practical solution for environmental monitoring.

References

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3. Standing Committee of Analysts. The determination of steroid oestrogens in waters using chromatography and mass spectrometry. 2008.
4. EU Water Framework Directive. http://ec.europa.eu/environment/water/water-framework/index_en.html