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

Monitoring trace levels of endocrine disrupting compounds is crucial to protect aquatic ecosystems and drinking water resources. 17alpha ethynylestradiol exhibits high estrogenic potency and is regulated under the EU Water Framework Directive with a detection limit of 35 pg/L.

Objectives and Study Overview

This study evaluates the feasibility of combining on-line solid phase extraction with high resolution Orbitrap mass spectrometry to achieve rapid quantitation and confirmation of 17alpha ethynylestradiol at or below regulatory limits in wastewater.

Methodology and Instrumentation

Online SPE was performed using the EQuan MAX Plus system with a 5 mL sample volume loaded at 0.5 mL per minute onto an Accucore aQ loading column. Analytical separation used an Acclaim Vanquish Polar Advantage column with mobile phases of ammonium fluoride in water and methanol. Detection employed a Q Exactive Focus quadrupole-Orbitrap mass spectrometer with heated electrospray ionization in negative mode, operating in parallel reaction monitoring at 70 000 resolving power. Data acquisition and processing used TraceFinder 4.1 software.

Main Results and Discussion

The method achieved a limit of detection of 15 pg/L and a limit of quantitation of 29 pg/L with a relative standard deviation of 3.4 percent. Calibration showed excellent linearity (R2 0.9998) and mass errors below 1 ppm. Total analysis time was under 30 minutes, approximately 30 times faster than conventional workflows. Application to real wastewater samples yielded confirmed detection of 17alpha ethynylestradiol at 462 pg/L and indications of chromatographic resolution of the 17beta isomer.

Benefits and Practical Applications

The rapid on-line SPE and high resolution detection reduce analysis time, solvent consumption, and operational costs while ensuring reliable quantitation and confirmation. This approach supports routine monitoring for regulatory compliance and environmental risk assessment.

Future Trends and Potential Applications

Further enhancements may include the use of stable isotope labelled internal standards to improve accuracy and extension of the method to other steroid estrogens such as estrone and 17beta estradiol. Integration into automated monitoring networks could enable high throughput surveillance of endocrine disruptors in water systems.

Conclusion

The combination of on-line SPE and Orbitrap mass spectrometry provides a fast, sensitive, and robust workflow for detecting 17alpha ethynylestradiol at sub regulatory levels in wastewater matrices.

Reference

• Thrupp TJ Runnalls TJ Scholze M Kugathas S Kortenkamp A Sumpter JP The consequences of exposure to mixtures of chemicals Something from nothing and a lot from little when fish are exposed to steroid hormones Sci Total Environ 2018 611 619 620
• Runnalls TJ Margiotta Casaluci L Kugathas S Sumpter JP Pharmaceuticals in the aquatic environment Steroids and anti steroids as high priorities for research Hum Ecol Risk Assess 2010 19 1318 1338
• Standing Committee of Analysts The determination of steroid oestrogens in waters using chromatography and mass spectrometry Report 2008
• EU Water Framework Directive European Union Water Framework Directive environmental standard