Bioanalytics – Chemical
2019 PharmSci 360
Lack of authentic matrices for external calibration curves is one of the challenges in LC-MS/MS biomarker analysis. Without the need of using external calibration curves, a novel methodology of In-Sample Calibration Curves (ISCC) using Multiple Isotopologue Reaction Monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a SIL-analyte for instant LC-MS/MS bioanalysis and quantitative proteomics will be presented.
The isotopic abundances of a SIL-analyte in its MIRM channels can be accurately calculated based on the fragmentation. The isotopic abundances can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of the SIL-analyte into each sample, an ISCC can be established based on the relationship between the calculated isotopic abundances (analyte concentration equivalents) and the measured LC-MS/MS peak areas in the corresponding MIRM channels of the SIL-analyte in each sample for the quantative analysis.
The MIRM-ISCC-LC-MS/MS methodology addresses the key issues and challenges in current LC-MS/MS bioanalysis, and allows accurate bioanalysis for the first time without using external calibration curves, impacting the bioanalysis of biomarkers, biotherapeutics and small-molecule compounds. The methodology is especially useful for the following aspects: 1) biomarker measurement and targeted quantitative proteomics where authentic matrices are not available for external calibration curves; 2) LC-MS/MS assays in clinical diagnostic laboratories where higher throughput and faster data turnaround could be achieved by eliminating the external calibration curves; 3) LC-MS/MS bioanalysis of fresh frozen tissue biopsies, FFPE tumor tissues, or dried blood spot (DBS) samples where external calibration curve preparation is cumbersome, too expensive (because of the rare matrices) or impossible. This methodology will be discussed with two case studies, including critical considerations for developing accurate and robust MIRM-ISCC-LC-MS/MS assays.