Rapid determination of ethinylestradiol (17α-EE2) to 15 pg/L using EQuan MAX online SPE coupled to a Q Exactive Focus Orbitrap LC/MS/MS system

Significance of the topic

17α-Ethinylestradiol (17α-EE2) is one of the most potent endocrine disrupting compounds found in aquatic environments. Its low environmental concentration and strong estrogenic activity pose risks to freshwater ecosystems and drinking water quality. The EU Water Framework Directive requires a limit of detection of 35 pg/L for 17α-EE2, creating a significant analytical challenge for trace-level quantitation and confirmation.

Objectives and study overview

The primary goal was to evaluate an integrated online solid phase extraction (SPE) and high-resolution mass spectrometry workflow using the Thermo Scientific EQuan MAX Plus system coupled to a Q Exactive Focus Orbitrap. The study aimed to achieve the EU WFD detection limit for 17α-EE2, ensure reliable confirmation, and streamline analysis time compared to traditional offline methods.

Methodology and instrumentation

The workflow combined online SPE of 5 mL water samples with reversed-phase loading onto Hypersil GOLD aQ sorbent, followed by chromatographic separation on an Acclaim VANQUISH Polar Advantage column. A loading gradient and analytical gradient delivered separation in under 30 minutes. Detection was performed in negative electrospray ionization using Parallel Reaction Monitoring (PRM) at 70,000 resolving power. Fragment ions at m/z 143.0502 and 145.0660 were used for quantitation and confirmation, with a mass tolerance of 2 ppm.

Used Instrumentation

• Thermo Scientific EQuan MAX Plus online SPE LC/MS system with binary and quaternary pumps, autosampler, and column compartment
• Thermo Scientific Q Exactive Focus hybrid quadrupole-Orbitrap mass spectrometer with HESI-II source
• Hypersil GOLD aQ 20 × 2.1 mm SPE column and Acclaim VANQUISH Polar Advantage 150 × 2.1 mm analytical column

Main results and discussion

External calibration over 25–1600 pg/L yielded excellent linearity (R2 = 0.9998) and calibrant mass errors below 1 ppm. Six replicate analyses of a 100 pg/L standard produced a relative standard deviation of 3.4%. The calculated limit of detection was 15 pg/L (4.65 × SD) and limit of quantitation was 29 pg/L (9 × SD). Analysis of treated wastewater effluent detected 17α-EE2 at 337 pg/L. A second chromatographic peak with identical MS2 fragments but different fragment ratios suggested partial resolution of the 17β-EE2 isomer, highlighting the method’s selectivity.

Benefits and practical applications

• Achieves EU WFD detection limit with high confidence using MS/MS confirmation
• Analysis time under 30 minutes, nearly 30× faster than traditional offline extraction
• Reduces sample handling, solvent use, and operational costs
• Potential to distinguish 17α- and 17β-EE2 isomers, improving quantitation accuracy

Future trends and opportunities

Advances in online SPE and high-resolution Orbitrap detection will support monitoring of a broader range of trace contaminants in water. Improved chromatographic phases may further separate stereoisomers and structural analogs. Integration with automated data processing and machine learning could enhance screening for emerging endocrine disruptors in environmental and drinking waters.

Conclusion

The combined EQuan MAX Plus online SPE and Q Exactive Focus workflow delivers robust quantitation and confirmation of 17α-EE2 at sub-35 pg/L levels in under 30 minutes. The method meets EU regulatory requirements, reduces time and resource demands, and offers the potential to resolve isomeric interferences. This approach represents a significant advancement for routine environmental monitoring of potent endocrine disruptors.

References

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2. Runnalls T J et al Pharmaceuticals in the aquatic environment: steroids and anti-steroids as high priorities for research Hum Ecol Risk Assess 2010 16 1318-1338
3. Standing Committee of Analysts The determination of steroid oestrogens in waters using chromatography and mass spectrometry 2008 UK Government publication
4. EU Water Framework Directive ec europa eu environment water water-framework index en html