Determination of Endocrine-Disrupting Chemicals in Drinking Water at Sub ng/L Levels Using the Agilent 6495 Triple Quadrupole Mass Spectrometer

Significance of the Topic

The presence of endocrine-disrupting chemicals (EDCs) in drinking water at sub-ng/L levels raises significant human and environmental health concerns.
Regulatory agencies such as the US EPA enforce stringent limits (EPA Methods 539, 1698) to protect aquatic ecosystems and public health.
The ability to rapidly and reliably detect trace hormones without laborious sample preparation is critical for routine water quality monitoring.

Objectives and Study Overview

This study demonstrates a streamlined liquid chromatography–tandem mass spectrometry (LC-MS/MS) workflow using the Agilent 6495 Triple Quadrupole system in dynamic multiple reaction monitoring (DMRM) mode with fast polarity switching.
The main goals were to:

• Achieve sub-ng/L detection limits for seven key hormones regulated under EPA Method 539.
• Avoid time-consuming sample enrichment (solid phase extraction) by employing large-volume direct injection of tap water.
• Validate method linearity, precision, and accuracy for routine environmental analysis.

Methodology and Instrumentation

Reagents and Standards

• EPA 539 stock standard containing androstenedione, equilin, 17β-estradiol, estriol, estrone, 17α-ethinylestradiol, and testosterone.
• Working standards prepared by dilution in methanol:water (1:1 v/v) and spiked into tap water (Santa Clara, CA) at 0.1–10 ng/L.
• Solvent A: 0.4 mM ammonium fluoride in water; Solvent B: methanol:acetonitrile (1:1 v/v).

Chromatographic Conditions

• Column: Agilent Poroshell Phenyl Hexyl (2.1×100 mm, 2.7 µm).
• Flow rate: 0.4 mL/min; column temperature: 40 °C.
• Gradient: 1 % B to 95 % B over 12 min; 900 µL injection volume with draw/eject at 1 000 µL/min.

Used Instrumentation

• Agilent 1260 Infinity HPLC: binary pump, autosampler with 900 µL loop, 0 °C sample cooler, thermostatted column compartment.
• Agilent 6495 Triple Quadrupole LC/MS: JetStream ion source with dual-stage ion funnel and hexabore capillary, curved collision cell, high-voltage dynode detector.
• MassHunter software for data acquisition, source optimization, and quantitative processing.

Main Results and Discussion

The redesigned 6495 system delivered 2- to 5-fold signal enhancements compared to its predecessor.
Instrument detection limits (IDLs) ranged from 0.02 to 0.78 ng/L (RSD < 15 %), enabling clear differentiation from background noise.
Lower limits of quantitation (LLOQs, S/N > 10, RSD < 20 %, 80–120 % accuracy) matched IDL performance and supported quantitation down to 0.1 ng/L for most analytes.
Calibration curves over two orders of magnitude exhibited excellent linearity (R2 > 0.994) with 1/x weighting.
Overlayed MRM chromatograms showed baseline separation of all seven hormones within a 16 min run time.

Benefits and Practical Applications of the Method

• Elimination of solid phase extraction reduces sample preparation time, solvent use, and potential analyte loss.
• Large-volume direct injection simplifies workflow and increases laboratory throughput.
• Fast polarity switching and DMRM maximize instrument duty cycle, improving sensitivity and selectivity.
• Method robustness supports routine monitoring of municipal and environmental water supplies for trace EDC contamination.

Future Trends and Opportunities

Advancements in ion optics and detector design will continue to push detection limits lower and further simplify workflows.
Integration of automated sample handling and real-time data processing could enable on-site monitoring of drinking water.
Expanding the analyte panel to include additional emerging contaminants will enhance comprehensive water quality assessment.

Conclusion

The Agilent 6495 Triple Quadrupole LC/MS method offers a fast, sensitive, and reliable approach for quantitating trace hormones in drinking water without extensive sample enrichment.
Sub-ng/L detection limits, excellent precision, and linearity ensure compliance with regulatory requirements and support high-throughput environmental analysis.

References

1. Flores-Valverde A.M., Horwood J., Hill E.M. Disruption of the steroid metabolome in fish caused by exposure to the environmental estrogen 17α-ethinylestradiol. Environ. Sci. Technol. 2010, 44(9), 3552–3558.
2. Jenkins R. et al. Identification of androstenedione in a river containing paper mill effluent. Environ. Toxicol. Chem. 2001, 20(6), 1325–1331.
3. US EPA Method 539. Determination of Hormones in Drinking Water by SPE and LC-ESI-MS/MS. 2010.
4. US EPA Method 1698. Steroids and Hormones in Water, Soil, Sediment, and Biosolids by HRGC/HRMS. 2007.
5. Hindle R. Improved Analysis of Trace Hormones in Drinking Water by LC/MS/MS (EPA 539) using the Agilent 6460 Triple Quadrupole LC/MS. Agilent Technologies, 2013.