Mass Spectrometry Analysis of Hormones in Water by Direct Injection

Significance of the Topic

Hormones are recognized as emerging contaminants in water due to their endocrine-disrupting effects at sub-ng/L levels. Regulatory agencies including the US EPA and the EU have added several synthetic and natural hormones to monitoring and candidate contaminant lists. Reliable detection of these compounds in surface and drinking water is critical for assessing potential health risks and informing treatment requirements.

Objectives and Study Overview

This study aimed to develop and validate a fast, robust direct-injection LC-MS/MS method for quantifying eight hormones in tap water. Target analytes included 17-α-ethinylestradiol, 17-β-estradiol, estriol, estrone, equilin, 4-androstene-3,17-dione, progesterone, and testosterone. By using only 100 µL sample injections and eliminating large-volume extraction, the method seeks to maximize throughput, minimize handling errors, and achieve ng/L level sensitivity.

Methodology and Instrumentation

Sample Preparation and Standards

• Tap water matrix fortified with hormone mix at 1–500 ng/L.
• Individual stocks (1 µg/mL) prepared in acetonitrile or methanol; dilutions made for calibration.

Instrumentation Used

• Agilent 1290 Infinity II UHPLC with binary pump, autosampler, and thermostatted column compartment.
• Agilent InfinityLab Poroshell 120 EC-C18 column (150 × 2.1 mm, 2.7 µm).
• Agilent 6470 Triple Quadrupole MS with electrospray ionization and polarity switching.

Chromatographic and MS Conditions

• Mobile phase A: acetonitrile; B: 1 mM NH4F in water.
• Gradient from 30 % to 100 % A over 10 min; total run time 11 min; flow rate 0.3 mL/min; injection 100 µL.
• Optimized source parameters: capillary voltage 3000 V; nozzle voltage 0 V (positive mode), 500 V (negative mode); sheath gas 350 °C, 11 L/min; drying gas 250 °C, 10 L/min; nebulizer 45 psi.
• Dynamic MRM transitions selected for quantifier and qualifier ions; cell acceleration voltage 7 V; delta EMV adjustments for each polarity.

Main Results and Discussion

Chromatographic separation of eight hormones was achieved within 11 min with distinct retention times. Source optimizer tools revealed positive-ion compounds (keto-containing hormones) and negative-ion compounds (hydroxyl-rich steroids) required different capillary and nozzle voltages for maximal sensitivity. Incorporation of 1 mM ammonium fluoride improved negative-mode ionization. Calibration curves over 1–500 ng/L exhibited excellent linearity (r2 > 0.998) with intraday and interday precision and accuracy below 15 %. Method detection limits ranged from 0.1 ng/L (progesterone, testosterone) to 20 ng/L (17-α-ethinylestradiol).

Benefits and Practical Applications

This direct-injection approach streamlines analysis by removing solid-phase extraction steps, reducing sample handling and consumable costs. The rapid 11 min cycle time allows high throughput screening of drinking and surface waters. Sensitivity at sub-ng/L levels supports regulatory monitoring and research on hormone occurrence and fate.

Future Trends and Applications

To achieve even lower detection thresholds, future work could integrate online SPE for preconcentration or expand the method to additional emerging endocrine disruptors. High-throughput automation, coupling with high-resolution mass spectrometry, and application to complex matrices such as wastewater effluents represent promising directions for environmental monitoring.

Conclusion

The developed direct-injection LC-MS/MS method using the Agilent 1290 Infinity II and 6470 Triple Quadrupole provides a fast, sensitive, and reliable tool for quantifying eight hormones in water at ng/L levels. Its simplicity and robustness make it suitable for routine environmental screening and regulatory compliance testing.

References

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2. Ting Y.F.; Praveena S.M. Sources, Mechanisms, and Fate of Steroid Estrogens in Wastewater Treatment Plants: A Mini Review. Environmental Monitoring and Assessment 2017, 189(4), 178.
3. Kidd K.A.; Blanchfield P.J.; Mills K.H.; Palace V.P.; Evans R.E.; Lazorchak J.M.; Flick R.W. Collapse of a Fish Population after Exposure to a Synthetic Estrogen. Proceedings of the National Academy of Sciences 2007, 104(21), 8897.
4. 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 Application Note 2013, 5991-2473EN.