Determination of Hormones in Drinking Water by LC/MS/MS Using an Agilent Infi nityLab Poroshell HPH Column (EPA 539)

Importance of the Topic

Endocrine-disrupting hormones in drinking water pose a significant public health concern due to their biological activity at trace levels. Accurate monitoring of these compounds is essential to ensure water safety and regulatory compliance. EPA Method 539 targets seven key hormones, demanding high sensitivity and chromatographic stability under alkaline conditions.

Objectives and Study Overview

This work aimed to develop and validate an efficient LC/MS/MS method for simultaneous determination of seven hormones in finished drinking water, following EPA Method 539. Key goals included optimizing sample extraction, achieving low detection limits, and demonstrating column stability at high pH.

Methodology and Instrumentation

Sample preparation

• Solid phase extraction using Agilent SPEC 47 mm C18AR discs
• Conditioning with methanol and water, loading 1 L sample, washing, and elution with methanol
• Evaporation under nitrogen, reconstitution in 1 mL of 50% methanol, and centrifugation

Chromatography

• Columns: Agilent InfinityLab Poroshell HPH-C18, 3.0 × 100 mm, 2.7 μm and 4 μm
• Mobile phase A: 0.02% ammonium hydroxide in water (pH 10.5); B: 0.02% ammonium hydroxide in methanol
• Gradient from 50:50 to 10:90 B over 10 min; flow rate 0.4 mL/min; column at 25 °C; injection 20 μL

Mass spectrometry

• Agilent 6460 Triple Quadrupole LC/MS with electrospray ionization in positive and negative modes
• Multiple reaction monitoring for each analyte and isotope-labeled internal standards
• Gas temperature 300 °C; sheath gas 375 °C; nebulizer 25 psi; capillary 4000–4500 V

Instrumentation Used

• Agilent 1290 Infinity LC
• Agilent 6460 Triple Quadrupole MS
• Agilent SPEC 47 mm C18AR disc SPE and holder
• Agilent InfinityLab Poroshell HPH-C18 columns
• Eppendorf Minispin Plus centrifuge
• Digital vortex mixer

Main Results and Discussion

Linearity and Detection Limits

• Calibration range 0.5–70 ng/L with R² ≥ 0.996 for all hormones
• Method detection limits between 0.03 and 0.7 ng/L, meeting or exceeding EPA 539 criteria

Recovery and Precision

• Recoveries 82.6–105.6% with RSDs of 2.7–6.7% at fortified levels

Column Comparison and Stability

• 2.7 μm column delivered higher signal-to-noise and lower LODs than 4 μm, though both complied with method requirements
• HPH-C18 (2.1 × 100 mm, 2.7 μm) retained efficiency and retention factor after >6000 mL of pH 10.5 mobile phase

Practical Benefits and Applications

• Reliable trace-level hormone analysis for water quality monitoring and regulatory compliance
• Short run times and reduced solvent use enhance throughput and sustainability
• High-pH column durability lowers maintenance costs and downtime

Future Trends and Potential Applications

Extending this workflow to other endocrine disruptors and complex matrices can broaden environmental surveillance. Integration with high-resolution MS and novel SPE sorbents may further improve selectivity, sensitivity, and sample throughput.

Conclusions

The validated LC/MS/MS approach using Agilent Poroshell HPH-C18 columns and SPE provides sensitive, precise, and robust quantification of seven hormones in drinking water. High-pH chromatographic stability and routine instrument configurations support its adoption in regulatory and research laboratories.

References

1. EPA Method 539 – Determination of Hormones in Drinking Water by SPE and LC-ESI-MS/MS, EPA 815-B-10-001, November 2010
2. R. Hindle. Improved Analysis of Trace Hormones in Drinking Water by LC/MS/MS (EPA 539) using the Agilent 6460 Triple Quadrupole LC/MS; Agilent Technologies Application Note 5991-2473EN, 2013