Improving the Detection of Thyroglobulin in Human Plasma for Clinical Research by Combining SISCAPA Enrichment and Microflow LC-MS

Significance of the Topic

Sensitive and accurate measurement of thyroglobulin (Tg) in human plasma is critical for clinical research because conventional immunoassays can yield false negatives due to anti-Tg autoantibodies. LC-MS methods with peptide immunoenrichment offer improved selectivity and quantitation at low concentration levels while consuming less sample.

Objectives and Study Overview

This study evaluated a combined SISCAPA enrichment and microflow LC-MS approach using a dual-pump trapping ionKey/MS system. Goals included lowering the limit of quantitation (LLOQ) for Tg, reducing sample volume requirements, and maintaining accuracy, precision, and throughput relative to standard flow methods.

Methodology and Used Instrumentation

• Sample preparation: tryptic digestion of plasma, immuno-enrichment of a signature Tg peptide (FSPDDSAGASALLR) with stable isotope-labeled standard and magnetic antibody beads.
• Chromatography and detection: ACQUITY UPLC M-Class system with dual-pump trapping; ionKey/MS coupled to Xevo TQ-S in MRM mode; iKey Peptide BEH C18 separation device; MassLynx and TargetLynx software.
• Operating conditions: 20 µL injection, 3 µL/min microflow, trap loading at 50 µL/min, total cycle time 6.75 min, sub-2 ng/mL quantitation with 50 µL plasma.

Main Results and Discussion

A calibration curve from 2,000 to 0.64 amol on column showed excellent linearity and reproducibility (CV ~16% at 13 amol). Plasma titration demonstrated linear response down to 40 µL sample. In a head-to-head comparison with standard flow (Agilent 1290/6490), the microflow method achieved R2=0.998 correlation, superior precision, and agreement within 95% confidence by Bland-Altman analysis. Reverse curve experiments indicated an LLOD of 15 amol and an LLOQ of 45 amol on-column. Spiking purified Tg in bovine plasma yielded an overall assay LLOQ of ~0.78 ng/mL using 50 µL sample.

Benefits and Practical Applications

• Quantitation below 1 ng/mL Tg with 10× less plasma than standard flow.
• Improved precision across replicates and high analytical selectivity.
• Reduced solvent consumption and smaller injection volumes.
• Cycle time comparable to standard methods with similar throughput.

Future Trends and Possibilities

Wider adoption of microflow LC-MS with immunoenrichment may enable multiplexed biomarker panels in clinical research, further reduction in sample and solvent consumption, and integration with automated workflows for high-throughput precision diagnostics.

Conclusion

The dual-pump trapping ionKey/MS system combined with SISCAPA enrichment provides a robust and sensitive platform for Tg analysis, achieving sub-ng/mL quantitation using minimal plasma volumes while preserving accuracy, enhanced precision, and efficient throughput, making it a viable solution for clinical research.

References

1. Lame ME, Chambers EE. Improving a High Sensitivity Assay for the Quantification of Teriparatide in Human Plasma Using the ionKey/MS System. Waters Application Note, 720004948EN. 2015.