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

Significance of the topic

Thyroglobulin (Tg) serves as a key biomarker for thyroid function and cancer surveillance. Conventional immunoassays can be compromised by anti-Tg autoantibodies that interfere with antibody binding, yielding falsely low or non-quantifiable results. The combination of Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA) and LC-MS/MS overcomes this limitation by targeting a proteotypic Tg peptide after digestion, providing enhanced specificity and reliability for clinical research applications.

Objectives and Study Overview

The study aimed to develop and validate an automated SISCAPA LC-MS/MS workflow for quantitative measurement of Tg in human serum at low concentration (down to 0.1 ng/mL) using an Andrew+ Pipetting Robot. Key goals included assessment of analytical sensitivity, precision, matrix effects and method comparability with external quality assessment (EQA) materials.

Methodology and Used Instrumentation

The automated workflow integrates sample digestion, peptide capture and targeted LC-MS/MS analysis under OneLab control.

• Sample Preparation: 250 µL serum was denatured, reduced and trypsin-digested, followed by protease inhibition and capture of the surrogate peptide FSPDDSAGASALLR using anti-peptide magnetic beads.
• Internal Standard: 13C6,15N4-labelled FSP peptide ensured compensation for matrix effects and procedural variability.
• Automation Platform: Andrew+ Pipetting Robot with OneLab software enabled high-throughput, reproducible handling of 96-well plates.
• Chromatography: ACQUITY UPLC I-Class FL system coupled to an XSelect Premier HSS T3 column (2.1×50 mm, 2.5 µm) using a 2.6 min gradient with 0.01% formic acid and acetonitrile.
• Mass Spectrometry: Xevo TQ Absolute mass spectrometer operated in MRM mode monitoring transitions 704.0→586.9 and 704.0→687.4 for the analyte and 709.0→591.9 for the internal standard.

Main Results and Discussion

Functional sensitivity experiments (n=30 per level, three runs) demonstrated a signal-to-noise ratio >10:1 and precision <20% CV at 0.1 ng/mL Tg. QC precision across three concentration levels (0.3, 3, 35 ng/mL) yielded total reproducibility and repeatability ≤9.5% CV. Matrix effect assessments in six individual human serum samples showed peak area variation of 79–104% (RSD <9%) and analyte/ISTD response ratios of 96–105% (RSD <4%), confirming robust internal standard compensation. Comparison with 38 UK NEQAS samples revealed an initial proportional bias (Passing-Bablok y=0.2149+0.5861x, Bland-Altman –40.1%). Reassignment of calibration to the EQA All Laboratory Trimmed Mean reduced bias to –5.5% with regression y=–0.261+0.9384x.

Benefits and Practical Applications

This automated SISCAPA LC-MS/MS assay delivers highly specific Tg quantification unaffected by anti-Tg antibodies, with a low-volume requirement (250 µL) and capacity for sample archiving and reanalysis. The workflow’s precision and traceability make it suitable for clinical research, assay validation, QA/QC laboratories and studies requiring accurate Tg monitoring.

Future Trends and Potential Applications

Advances may include full integration into clinical diagnostic platforms, expansion to multiplexed peptide assays for thyroid and endocrine markers, refined calibration with traceable reference materials and adaptation to decentralized LC-MS laboratories. Emerging automation technologies and improved data analytics will further enhance throughput, standardization and clinical utility.

Conclusion

An automated SISCAPA LC-MS/MS method was developed for serum thyroglobulin quantification, achieving a functional sensitivity of 0.1 ng/mL and robust performance (≤9.5% CV). The approach circumvents antibody interference, demonstrates strong agreement with external quality standards after calibration adjustment and offers a scalable solution for clinical research laboratories.