Topic: Tox / TDM / Endocrine
Authors: Jennifer Kemp (1), Anthony Maus (1), Ravinder Singh (1) and Stefan Grebe (1,2)
For some analytes, such as thyroglobulin (TG), analytical sensitivity can be a challenge in clinical laboratory mass spectrometry (MS) testing. Tg testing performed by mass spectrometry has become an important disease-monitoring tool in thyroid cancer patients with anti-thyroglobulin autoantibodies (TGAB). TG measurement by a MS assay allows for accurate quantitation of TG in samples that contain TGAB interferences, which confound accurate quantification in immunoassays, through measurement of TG-specific tryptic peptides. Due to the need for monitoring low levels of circulating TG in such patients, we evaluated the sensitivity benefits of using microliter liquid chromatography (LC) flow rates by directly comparing the widely used Shimadzu 20 series pumps, paired with a Cohesive Technologies autosampler, operated at a flowrate of 300 mcL/min, with the Sciex M3 micro-flow LC system, using a 10 mcL/min flow rate.
Serum sample extracts, standards and quality controls from clinical testing were pooled to obtain results for the comparison. A standard curve and four quality control pools were run over the course of five days on both LC platforms to obtain imprecision. Fifteen patient pools, each consisting of three patient extracts, were injected daily to obtain accuracy. Extracts were prepared by precipitating high molecular weight proteins (>80 kDa), which included immunoglobulins and TG, with a solution of saturated ammonium sulfate. The pellets were re-suspended, reduced and alkylated. Stable isotope-labeled (13C) internal standard peptide (IS) was added, and the specimens were trypsin-digested for 16 h. Antibody-coated MSIA D.A.R.T.’S were used to capture the proteotypic TG peptide (FSPDDSAGASALLR), which was assayed post elution by multiple reaction monitoring (MRM) LC-MS/MS (SCIEX 6500+).
The calculated concentration results from both LC platforms showed good correlation with an r^2 of 0.986 and a slope of 1.008. The microflow LC system produced a 5.95 fold increase in signal-to-noise ratio. Imprecision was also improved when using the lower flow rate system, with an average inter day %CV of 3.3% for the M3 compared to 6.1% for the traditional flow rate.
The clinical need for high sensitivity for the TG assay can be best met by a low flow microLC system. This yielded substantial improvement in the signal-to-noise ratio and improved imprecision. In combination this led to a >5-fold lower limit of quantification, which improves the clinical utility of the test.
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