Quantitative Comparison of Hormones in Drinking Water Between MS/MS and Orbitrap Technology

Significance of the Topic

Endocrine disrupting compounds (EDCs) such as natural and synthetic hormones are increasingly detected in drinking water at trace levels. Their presence poses risks to human health and aquatic ecosystems by interfering with normal hormonal functions. Regulatory agencies, including the US EPA, have established monitoring guidelines (EPA Method 539) to track these micropollutants. Advanced, high‐resolution mass spectrometry techniques can improve sensitivity and selectivity, ensuring compliance with stringent reporting limits and enhancing public health protection.

Objectives and Study Overview

This study aims to compare the quantitative performance of two mass spectrometric approaches for hormone analysis in drinking water according to EPA Method 539:

• Triple quadrupole MS/MS using selected reaction monitoring (SRM/MRM).
• High‐resolution accurate mass (HRAM) Orbitrap technology employing parallel reaction monitoring (PRM).

The goal is to demonstrate whether the Orbitrap‐based LC‐HRAM method can match or exceed the sensitivity, precision, and accuracy of the established triple quadrupole workflow while offering additional flexibility for compound identification and non-targeted screening.

Methodology and Instrumentation

Sample Preparation:

• 500 mL drinking water samples dechlorinated and spiked with surrogates.
• Solid phase extraction (C18) followed by concentration to dryness.
• Reconstitution in 50:50 methanol/water, addition of internal standards, and 50 µL injection.

Chromatography:

• Thermo Scientific UltiMate 3000 RS UHPLC with Acclaim PolarAdvantage II column (2.1 × 150 mm, 3 µm).
• Gradient elution at 0.3 mL/min using 1 mM ammonium fluoride in water and acetonitrile/methanol (50:50).

Mass Spectrometry:

• Triple quadrupole MS in SRM mode (EPA Method 539 settings).
• Thermo Q Exactive Quadrupole‐Orbitrap MS in PRM mode.
• Orbitrap resolving power of 70 000 FWHM, AGC target 2 × 10⁵, maximum injection time 200 ms, isolation window 1.0 m/z.

Key Results and Discussion

• Linearity and Detection Limits:
Both SRM and PRM methods showed excellent calibration linearity down to one-fourth of the minimum reporting level (MRL). PRM achieved lower critical levels and calculated detection limits than those reported for EPA 539 triple quadrupole analyses.
• Precision and Accuracy:
Replicate analyses (n = 4) in fortified reagent water and two UCMR3 matrices demonstrated recoveries between 81 % and 118 % with relative standard deviations ≤ 8 %, meeting EPA IDC criteria.
• Method Robustness:
The LC-HRAM approach provided consistent performance across different water matrices, confirming its reliability for trace hormone quantification.

Benefits and Practical Applications

• Comparable Quantitation: Orbitrap PRM matches or exceeds triple quadrupole sensitivity and precision for regulated hormones.
• Enhanced Selectivity: High-resolution MS allows unambiguous fragment ion assignment and reduces interference from complex matrices.
• Flexibility: The same PRM acquisition can support targeted quantitation, full‐scan screening, and retrospective data mining without additional runs.
• Regulatory Compliance: Meets EPA Method 539 requirements for reporting limits, precision, and accuracy.

Future Trends and Opportunities

• Non-targeted and Suspect Screening: Integrating HRAM with advanced data analysis workflows to detect emerging contaminants beyond the target list.
• Multi-class Analysis: Expanding the method to include pharmaceuticals, personal care products, and other micropollutant groups in a single run.
• Automation and Miniaturization: Streamlining sample preparation and reducing solvent use for high-throughput monitoring.
• Real-time Monitoring: Developing online SPE-HRAM systems for near real-time water quality assessment.

Conclusion

The LC-HRAM methodology using Orbitrap technology offers a sensitive, accurate, and reproducible alternative to conventional triple quadrupole MS for hormone quantification in drinking water. By fulfilling EPA Method 539 criteria and enabling combined targeted and non-targeted analysis, the high-resolution approach enhances analytical flexibility and bolsters routine compliance monitoring.

References

1. US EPA. Contaminants of Emerging Concern Including Pharmaceuticals and Personal Care Products. EPA Water Quality Center. 2016.
2. Winslow SD et al. Statistical Procedures for Determination and Verification of Minimum Reporting Levels for Drinking Water Methods. Environ. Sci. Technol. 2006, 40, 281–288.
3. US EPA Method 539: Determination of Hormones in Drinking Water by SPE and LC-ESI-MS/MS, v1.0 (2010).
4. US EPA Unregulated Contaminant Monitoring Rule 3 (UCMR3). 2016.
5. Richardson SD; Ternes TA. Water Analysis: Emerging Contaminants and Current Issues. Anal. Chem. 2014, 86, 2813–2848.