Sensitive Detection of Three Forms of Thyroid Hormone in Human Serum Using the Agilent 6490 Triple Quadrupole LC/MS System

Importance of the Topic

A sensitive and rapid analytical approach for quantifying free and total thyroid hormones in human serum is crucial for accurate assessment of thyroid function. Low circulating levels of free thyroxine (T4), triiodothyronine (T3) and reverse T3 (rT3) play key roles in endocrine health, and reliable measurements at picogram-per-milliliter concentrations support research, clinical diagnostics, and quality control in pharmaceutical and clinical laboratories.

Objectives and Study Overview

This study aimed to develop and validate a fast, robust LC/MS/MS method for simultaneous determination of free and total T4, T3 and rT3 in human serum. The method leverages multiple reaction monitoring (MRM) on an Agilent 6490 Triple Quadrupole LC/MS system, coupled with simple sample preparation protocols.

Methodology and Instrumentation

Sample Preparation:

• Free hormones: Ultrafiltration using Amicon YM-30 filters followed by protein precipitation with acetonitrile.
• Total hormones: Liquid-liquid extraction (LLE) with ethyl acetate after protein crash in acetonitrile.

Instrumentation:

• LC System: Agilent 1290 Infinity with Poroshell 120 EC-C18 column (3.0 × 100 mm, 2.7 µm).
• MS Detection: Agilent 6490 Triple Quadrupole with Jet Stream technology in positive ionization mode.
• Chromatography: 6.5 min gradient from 30% to 98% acetonitrile with 0.1% acetic acid, flow rate 0.3 mL/min at 20 °C.
• MRM Transitions: Optimized precursor/product ion pairs for each analyte and 13C-labeled internal standards; dwell times set at 50 ms.

Main Results and Discussion

Chromatographic separation achieved baseline resolution of T3, rT3 and T4 within a single 6.5-minute run. Calibration curves for both free and total analytes were linear over 0.5–1,000 pg/mL with R² ≥ 0.990. Lower limits of quantitation (LLOQ) were:

• Free T3: 1 pg/mL; Free T4 and rT3: 5 pg/mL.
• Total T3: 0.5 pg/mL; Total T4: 1 pg/mL; Total rT3: 2.5 pg/mL.

Method precision and accuracy met acceptance criteria, demonstrating suitability for quantifying trace thyroid hormones in complex serum matrices.

Benefits and Practical Applications

This LC/MS/MS workflow offers:

• High sensitivity enabling detection at low pg/mL levels.
• Rapid analysis with a 6.5-minute run time and minimal sample handling.
• Versatility for both free and total hormone measurements.
• Application in clinical research, diagnostic laboratories, and pharmaceutical quality control.

Future Trends and Potential Applications

Advancements may include:

• Multiplexing additional endocrine markers for comprehensive panels.
• Automation of sample preparation to increase throughput.
• Integration with high-resolution MS for enhanced specificity.
• Adaptation to microsampling formats for pediatric and remote testing.

Conclusion

The developed method provides a robust, sensitive, and efficient solution for simultaneous quantification of free and total thyroid hormones in human serum. Its strong linearity, low LLOQs, and fast throughput make it well suited for clinical and research applications requiring reliable low-level hormone measurement.

Reference

Agilent Application Note: Sensitive Detection of Three Forms of Thyroid Hormone in Human Serum Using the Agilent 6490 Triple Quadrupole LC/MS System, Agilent Technologies, Inc., 2014.