QUANTIFICATION OF THYROGLOBULIN IN SERUM FOR CLINICAL RESEARCH USING SISCAPA WORKFLOW COMBINED WITH LC-MS/MS

Importance of the Topic

Accurate measurement of thyroglobulin (Tg) in serum is critical for clinical research and monitoring thyroid disorders. Conventional immunoassays may be compromised by anti-Tg antibodies leading to underestimation of Tg levels. A robust LC-MS/MS approach combined with SISCAPA enrichment offers a way to overcome this interference, providing sensitive and specific quantification of Tg peptides in human serum samples.

Study Objectives and Overview

This study aimed to develop and automate a SISCAPA-based LC-MS/MS workflow for quantification of Tg in serum for clinical research applications. Key goals included achieving a functional sensitivity of 0.1 ng/mL from 250 µL serum, ensuring precision and reproducibility, evaluating matrix effects, and comparing performance against external quality assessment standards.

Methodology and Instrumentation

The assay employed proteolytic digestion of serum samples followed by immuno-affinity capture of a Tg-specific peptide surrogate (FSPDDSAGASALLR). Key steps:

• Sample preparation: 250 µL serum digested with denaturant, reducing agent, trypsin, and protease inhibitor on an Andrew+ Pipetting Robot.
• SISCAPA enrichment: automated capture using anti-peptide beads, magnetic separation, washing and elution.
• Internal standard: stable isotope-labeled FSP peptide added to all samples.
• LC-MS/MS analysis: separation on ACQUITY UPLC I-Class FL with XSelect Premier HSS T3 column (2.1×50 mm), 2.6-minute gradient; detection on Xevo TQ Absolute via MRM transitions for native and SIL peptides.

Main Results and Discussion

• Analytical Sensitivity: Functional sensitivity at 0.1 ng/mL with precision <20% and S/N >10:1.
• Reproducibility: Total CV ≤9.5% across QC levels (0.3, 3, 35 ng/mL) in surrogate and human serum over five runs.
• Matrix Effects: Peak area recovery 79–104% (RSD <9%), analyte/IS ratio 96–105% (RSD <4%), demonstrating robust internal standard compensation.
• EQA Comparison: Initial bias against UK NEQAS ALTM was –40.1%; recalibration of in-house standards to ALTM reduced bias to –5.5% and achieved near-unity regression (y=–0.261+0.9384x).

Benefits and Practical Applications

This automated SISCAPA LC-MS/MS method delivers high sensitivity and precision for Tg quantification while overcoming antibody interference. The low sample volume requirement and automation facilitate high-throughput workflows, supporting clinical research and potential QA/QC in specialized laboratories. The robust internal standard strategy ensures reliable data across diverse serum matrices.

Future Trends and Applications

• Extension to routine clinical diagnostics through further validation and regulatory approval.
• Multiplexed assays for simultaneous measurement of thyroid and other biomarkers.
• Enhanced automation and integration with laboratory information systems.
• Development of standardized calibrators traceable to international reference materials.

Conclusion

The developed SISCAPA LC-MS/MS workflow enables precise and sensitive quantification of thyroglobulin in serum, achieving 0.1 ng/mL detection from small volumes and demonstrating excellent reproducibility, matrix tolerance, and alignment with external quality standards after calibrator realignment. Automation on the Andrew+ platform supports scalability for clinical research applications.

References

No additional literature references were provided in the original text.