Sensitive assay of free thyroid hormones by on line SPE-UHPLC-MS/MS in human plasma

Significance of the Topic

The accurate quantification of free thyroid hormones, T3 and T4, in human plasma is critical for diagnosing and managing thyroid disorders. Only a fraction of circulating hormones exists in the unbound form, typically in the low picogram per milliliter range, yet these free levels directly reflect the hormonal activity at the tissue level. Ultra-sensitive and selective analytical methods are therefore essential to measure these low-abundance analytes reliably in clinical and research settings.

Objectives and Study Overview

This study aimed to develop and validate an on-line solid-phase extraction (SPE) coupled with ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the rapid and sensitive determination of free T3 and T4 in human plasma. Reverse T3 (rT3) was monitored to confirm the selectivity of T3 quantification but was not directly quantified due to isobaric interference.

Methodology and Instrumentation

Sample Preparation and SPE Workflow:

• Ultrafiltration of 500 µL plasma at 2000 g for 20 min to isolate free hormones.
• Injection of 120 µL ultrafiltrate spiked with 13C6-labeled internal standards (10 pg/mL) onto an on-line SPE column.
• Mixed-mode Phenomenex STRATA-XC (20×2 mm) column used for protein and matrix cleanup.
• Sequential SPE program: aqueous sample loading, organic wash for interferences, backflush into UHPLC, column cleaning and reconditioning.

UHPLC-MS/MS Separation and Detection:

• UHPLC on Synergi Fusion-RP C18 (50×2 mm, 2.5 µm) with gradient elution from 10 to 100% acetonitrile plus ammonia modifier, 0.4 mL/min, 30°C.
• Detection on Shimadzu LCMS-8040 triple quadrupole in negative ESI MRM mode.
• Key transitions: T3 649.70>126.90 (quantifier), 649.70>632.75 (qualifier); T4 775.50>126.90, 775.50>604.80; rT3 649.50>126.90, 649.50>604.80.

Used Instrumentation

• Millipore Centrifree ultrafiltration device.
• Shimadzu on-line SPE module and Nexera MP UHPLC system.
• Phenomenex STRATA-XC SPE and Synergi Fusion-RP UHPLC columns.
• Shimadzu LCMS-8040 triple quadrupole mass spectrometer.

Main Results and Discussion

The method achieved linear calibration from 1 to 50 pg/mL for both T3 and T4, with the lowest point at 1 pg/mL demonstrating clear signal-to-noise and retention time stability. Typical patient samples containing ~10 pg/mL hormones showed well-resolved peaks for T3, T4, and monitored rT3, confirming selectivity. The mixed-mode SPE effectively minimized matrix effects, and on-line cleanup accelerated sample throughput without compromising sensitivity.

Benefits and Practical Applications

• High sensitivity enabling reliable detection at low picogram levels.
• Automated on-line SPE reduces manual handling and risk of contamination.
• Short total analysis cycle (~8.5 min) supports high sample throughput.
• Applicability for clinical diagnostics, pharmacokinetic studies, and quality control of thyroid hormone therapies.

Future Trends and Potential Applications

Further miniaturization and integration with microfluidic platforms could reduce sample volume and analysis time. Expanding the analyte panel to include iodinated metabolites or conjugated forms may provide deeper insight into thyroid metabolism. Coupling with high-resolution mass spectrometry could enhance structural elucidation and isobaric separation.

Conclusion

The presented on-line SPE-UHPLC-MS/MS workflow delivers a robust, sensitive, and high-throughput assay for free T3 and T4 in plasma. It meets the stringent requirements for clinical research and diagnostic environments while providing precise quantification at low picogram concentrations.

Reference

• Ramero M, Moreau S, Levi M. Sensitive assay of free thyroid hormones by on line SPE-UHPLC-MS/MS in human plasma. ASMS 2013, WP-098.