Analysis of Estrone and 17β-Estradiol in Ground Water by GC-NCI-MS/MS

Importance of the Topic

The detection of trace levels of natural hormones such as estrone and 17β-estradiol in groundwater is critical due to their potent endocrine-disrupting effects on aquatic organisms. Even femtogram-level concentrations can induce reproductive abnormalities and feminization in fish. Developing highly sensitive and selective analytical methods is essential for environmental monitoring and regulatory compliance.

Objectives and Study Overview

This study aimed to establish a gas chromatography–negative chemical ionization–tandem mass spectrometry (GC-NCI-MS/MS) workflow for quantifying estrone (E1), 17β-estradiol (E2), and additional steroid hormones in groundwater. Key goals included achieving detection limits comparable or superior to existing LC-MS/MS methods while drastically reducing sample extraction volumes from 500 mL to as little as 20 mL.

Methodology and Derivatization

The hormones were derivatized to enhance volatility and ionization: E1 and E2 were converted into pentafluorobenzoyl (PFB) esters or PFB oximes, and estriol (E3) to its PFB oxime and trimethylsilyl (TMS) ether. Samples (20 mL groundwater) were spiked with a deuterated internal standard (TEST-D5 at 10 pg/mL), extracted, and concentrated. Calibration covered 0.05 pg/mL to 10 ng/mL.

Used Instrumentation

• Agilent 7890A Gas Chromatograph with Multi-Mode Inlet and back-flush configuration using a purged ultimate union.
• Two DB-17ht capillary columns: 5 m × 0.25 mm × 0.15 μm and 15 m × 0.25 mm × 0.15 μm.
• Agilent 7000B Triple Quadrupole Mass Spectrometer operating in negative chemical ionization (NCI) with 40% ammonia reagent gas at 150 °C.
• Selected reaction monitoring (SRM) transitions (e.g., E2 538→474 m/z, CE 3 V) for high specificity.

Main Results and Discussion

The GC-NCI-MS/MS method achieved on-column detection limits of 1.7 fg for E2 (0.05 pg/mL) and demonstrated excellent linearity (R2 > 0.998) across 5 pg/mL to 10 000 pg/mL. Precision, expressed as %RSD for five replicates of laboratory-spiked water at 0.5 pg/mL, was below 9%. Back-flush operation effectively eliminated carryover, improving cycle time and throughput. A blind proof-of-principle on EPA groundwater samples confirmed accuracy, with spike recoveries within 28% for most compounds using 20 mL extracts.

Benefits and Practical Applications

• Significant reduction in required sample volume (from 500 mL to 20 mL) lowers solvent usage and preparation time.
• High sensitivity and selectivity enable reliable quantification of trace estrogenic compounds in complex matrices.
• Back-flush-enabled workflow minimizes carryover, enhancing laboratory throughput and cost efficiency.
• Adaptable SRM method can be extended to other environmental contaminants such as androgens and progestins.

Future Trends and Potential Applications

Advances may include automated on-line extraction coupled to GC-NCI-MS/MS for real-time monitoring, integration with high-throughput environmental screening programs, and expansion of the method to a broader panel of endocrine-active compounds. Further miniaturization of sample preparation and multi-residue workflows will support large-scale water quality assessments.

Conclusion

The developed GC-NCI-MS/MS approach offers a robust, ultra-sensitive alternative to LC-MS/MS for hormone analysis in groundwater. By combining selective derivatization, negative chemical ionization, and back-flush chromatography, the method achieves femtogram-level detection, low carryover, and reduced sample requirements, making it a powerful tool for environmental monitoring.

References

1. Churley M, Macherone A, White R. Highly Repeatable Ultra Low Detection of Estradiol Using Triple Quadrupole GC/MS in NCI Mode. Application Brief 5990-5513EN. Agilent Technologies, Inc.; 2010.
2. Lee YH, Wu GC, Du JL, Chang CF. Estradiol-17β-induced a reversible sex change in the fingerlings of protandrous black porgie, Ancanthopagrus schlegeli. Biology of Reproduction. 71(5):1270-1278; 2004.
3. Bleeker W. The possible roles of luteinizing hormone in sex change. Biology of Reproduction. 71(5):1270-1278; 2004.
4. Macherone A, Churley M, White R. Ultra-low Detection of Estrogenic Compounds by GC-NCI/MS/MS. LC/GC Special Issues. Advanstar Communications, Inc.; 2010.