Reverse Triiodothyronine (rT3) Quantitation in Blood Serum for Research Purposes by LC-MS/MS using Liquid-Liquid Extraction following Protein Precipitation

Significance of the Topic

Reverse triiodothyronine (rT3) is an inactive isomer of the thyroid hormone triiodothyronine (T3). Quantifying rT3 in serum aids research into metabolic responses during starvation, critical illness and thyroid dysfunction. A reliable, sensitive LC-MS/MS method supports accurate biomarker measurement, contributing to insights in endocrinology and clinical chemistry.

Objectives and Study Overview

This study presents a six-minute LC-MS/MS research method for the quantitation of rT3 in human blood serum over the range of 5–60 ng/dL. The main goals were to:

• Develop a sample preparation workflow combining protein precipitation and liquid-liquid extraction.
• Achieve chromatographic separation of rT3 from T3 and other interferences.
• Validate precision, accuracy and throughput using a four-channel LC-MS/MS system.
• Compare results with those from a reference laboratory.

Methodology and Instrumentation

Sample preparation:

• Aliquots of serum were mixed with water, acetonitrile and 13C6-labeled rT3 internal standard.
• Following protein precipitation, ethyl acetate extraction removed matrix components.
• Extracts were dried and reconstituted in 25% acetonitrile.

Chromatography and detection:

• Thermo Scientific Transcend LX-4 UHPLC system with heated Accucore aQ column (100×2.1 mm, 60 °C).
• Gradient elution from 5% to 100% methanol in water (0.1% formic acid) over 6 minutes.
• Thermo Scientific TSQ Quantis triple-stage quadrupole MS/MS with heated electrospray ionization.
• Selected reaction monitoring transitions: 651.8→605.8 (quantifier), 651.8→507.9 (qualifier); internal standard transitions accordingly.

Used Instrumentation

• Transcend LX-4 UHPLC system.
• Accucore aQ 2.6 µm, 100×2.1 mm column at 60 °C.
• TSQ Quantis MS/MS with HESI source.
• TraceFinder software with Aria MX for system control and data analysis.

Main Results and Discussion

Precision and accuracy:

• Intra-batch precision: CV <6% for low, medium, high QC levels.
• Inter-batch precision: CV <8% across 22 days.
• Accuracy vs reference lab: mean bias +2%, with only one sample exceeding 20% difference.

Linearity and carryover:

• Linear response from 5 to 60 ng/dL with average ion ratio confirmation of 55%.
• Carryover <1% (<0.6 ng/dL) in blank injections following high calibrator.

Matrix effects and selectivity:

• Moderate ion suppression recovered by internal standard (average recovery 69%).
• Good separation from T3/T4; reliable quantitation in lipemic and icteric sera, but not in hemolyzed samples.

Benefits and Practical Applications

This method provides:

• High throughput of up to 36 injections per hour using four LC channels.
• Robust quantitation suitable for research on metabolic stress, thyroid disorders and nutritional studies.
• Compatibility with multiplexed LC-MS/MS assays under similar ion-source conditions.
• Low carryover and high reproducibility for longitudinal studies.

Future Trends and Possibilities

Emerging developments may include:

• Integration with automated sample handling and online extraction techniques.
• Expansion to dried blood spot or capillary microsampling for field research.
• Coupling with high-resolution mass spectrometry for enhanced selectivity.
• AI-driven data processing to accelerate method optimization and peak review.

Conclusion

The described LC-MS/MS protocol delivers accurate, precise and high-throughput quantitation of rT3 in serum. Its strong agreement with reference laboratory data and resilience to common matrix interferences make it valuable for metabolic and endocrinology research.