In-Sample Calibration Curve Using Multiple Isotopologue Reaction Monitoring of a Stable Isotopically Labeled Analyte for Instant LC-MS/MS Bioanalysis and Quantitative Proteomics

A novel methodology of in-sample calibration curves (ISCC) using multiple isotopologue reaction monitoring (MIRM) of multiple naturally occurring isotopologue transitions of a stable isotopically labeled (SIL) analyte for instant liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalysis of biomarkers, biotherapeutics, and small-molecule compounds is proposed and demonstrated for the first time. The theoretical isotopic abundances of the SIL analyte in its MIRM channels can be accurately calculated based on the isotopic distributions of its daughter ion and neutral loss. The isotopic abundances in these MIRM channels can also be accurately measured with a triple quadrupole mass spectrometer. By spiking a known amount of a SIL analyte into each study sample, an ISCC can be established based on the relationship between the calculated theoretical isotopic abundances (analyte concentration equivalents) in the selected MIRM channels of the SIL analyte and the measured MS/MS peak areas in the corresp...