B-167 Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS

Abstract Background Accurate measurement of Thyroglobulin (Tg) using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA™) Workflow combined with LC-MS/MS has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Methods Using a Andrew+™ Pipetting Robot, denaturation and reduction buffer was added to 250µL serum samples and incubated at 37°C. Internal standard and trypsin were added and samples incubated at 37°C. The samples were quenched with protease inhibitor and Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes. Using a magnetic array plate, the beads were left to pull down, samples were discarded, and the beads were twice washed with PBS/CHAPS prior to elution with acetonitrile with formic acid. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ HSS T3 Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (20%) at 0.1 ng/mL with S/N (PtP) 10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements. Conclusions A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.