Extraction of Thyroid Hormones: T3, rT3 and T4 from Serum Using EVOLUTE® EXPRESS AX Plates Prior to LC-MS/MS Analysis

Importance of the Topic

Serum levels of thyroid hormones such as T3, rT3 and T4 are critical biomarkers for diagnosing thyroid disorders. Accurate measurement of these analytes requires efficient sample cleanup to remove matrix components that interfere with LC-MS/MS analysis. A robust extraction method enhances sensitivity, reproducibility and assay reliability in clinical and pharmaceutical contexts.

Study Objectives and Overview

The aim of the study was to develop a selective strong anion exchange SPE protocol using a polymer-based EVOLUTE EXPRESS AX 30 mg fixed well plate for extracting T3, rT3 and T4 from minimal serum volume. The workflow was optimized to achieve high recovery, low phospholipid contamination and quantitation limits below 50 pg/mL for thyroid hormones.

Methodology and Instrumentation

• Sample Pretreatment: Serum (200 µL) spiked with deuterated internal standard and bulk stabilizers (citric acid, ascorbic acid, DL-dithiothreitol) to prevent in situ hormone conversion.
• SPE Protocol: Plate conditioning with methanol, equilibration with water, sample loading, sequential washes under controlled pH to remove interferences, and final elution with methanol.
• Postprocessing: Addition of ethylene glycol to minimize nonspecific adsorption, evaporation at 40 °C, reconstitution in water/methanol (50/50).
• Instrumentation: UPLC with ACE EXCEL2 C18 UHPLC column, Waters ACQUITY I-Class, flow rate 0.4 mL/min, column at 40 °C; Xevo TQ-S triple quadrupole mass spectrometer in MRM mode for targeted detection.

Main Findings and Discussion

• Chromatographic Separation: Baseline resolution of T3, rT3 and T4 achieved in under 3 minutes, minimizing in-source deiodination and cross-contamination.
• Recovery and Precision: Recoveries above 85% with RSDs below 10% across a spiking range of 25–2000 pg/mL.
• Calibration Performance: Linear response for T3 and rT3 from 25 to 1000 pg/mL (r2 > 0.99); T4 quantification impacted by endogenous background.
• Matrix Cleanliness: SPE protocol effectively removed phospholipids, enhancing assay robustness and reducing matrix effects.

Advantages and Practical Applications

The described method offers rapid, reproducible extraction suitable for high-throughput clinical and research laboratories. Its low sample volume requirement and strong cleanup capability make it ideal for pharmacokinetic, toxicology and thyroid disorder studies.

Future Trends and Opportunities

• Integration with automated platforms to increase throughput and reduce hands-on time.
• Extension to other endocrine biomarkers using tailored anion exchange sorbents.
• Development of multiplexed assays combining thyroid hormones with related metabolic markers for comprehensive profiling.
• Utilization of high-resolution mass spectrometry to further improve specificity and limit of detection.

Conclusion

The EVOLUTE EXPRESS AX SPE approach provides a reliable, efficient solution for extracting and quantifying T3, rT3 and T4 in serum. Its high recovery, low matrix interference and fast chromatographic analysis support its adoption in diverse analytical settings.

References

No external literature references were listed in the source document.