Human cardiac troponin I (cTnI) in serum is a well-known clinical biomarker for cardiac tissue damage and is used for diagnosing myocardial infarction. Unfortunately, commercial cTnI immunoassays from different manufacturers can produce significantly different measurement results for the same sample. In order to improve the comparability of these measurements, clinical cTnI immunoassays need to be standardized. Ultimately, the goal of this work was to develop an isotope dilution-liquid chromatography-mass spectrometry (ID-LC-MS/MS) reference measurement procedure that could be used to assign concentration values to cTnI reference materials. However, given that most serum protein biomarkers are low abundant, enrichment was mandatory to successfully quantify cTnI at clinically significant concentrations (≈ 1-10 ng/mL). As such, the specific aim of this work was to develop enrichment techniques and an ID-LC-MS/MS method to quantify cTnI in patient serum samples.

In order to achieved the required LC-MS/MS sensitivity, novel enrichment strategies were investigated to selectively isolate cTnI from serum and plasma. Silica coated magnetic nanoparticles were synthesized and conjugated with antibodies to act as immunoaffinity carriers. Magnetic nanoparticles were selected due to their variable surface modifications, high binding capacity, and the fact that they can be easily isolated using a magnet. After optimizing the enrichment and digestion procedures, isotopically labeled cTnI proteins were used as an internal standard for ID-LC-MS/MS analysis of cTnI to compensate for variations in the sample preparation. Finally, the developed LC-MS/MS-based assay was applied to measure cTnI concentrations in patient plasma samples. Effective enrichment methods proved to be crucial for achieving quantification of cTnI by ID-LC-MS/MS. To this end, a complementary ID-LC-MS/MS method was also developed to evaluate different antibody immobilization strategies and magnetic particle types as part of the method optimization. Overall, this work demonstrates significant improvements in magnetic particle enrichment techniques and LC-MS/MS detection for the analysis of cTnI in patient samples.