The key to understanding the IROA methodology is that we create both 12C and 13C isotopes to be uniformly present at approximately 5% for one isotope and approximately 95% for the second isotope. The molecules labeled at 5% 13C have a strongly enhanced M+1 and the molecules labeled at 95% 13C a strongly enhanced M-1, creating a mirror-image of one another (see Figure 1). Using traditional comprehensive (>98%) labeling, the monoisotopic peak of most compounds can usually be detected even if its intensity is low, but the M+1/M-1 minor peaks can be easily lost. Where the 13C is increased to 5% or 95%, the M+1 and M-1 peaks for a six-carbon molecule such as arginine in Figure 1 become significantly larger, namely 32% of the height of the monoisotopic peak. Whereas if the 13C is present at only 1.1%, the height of the M+1 is only approximately 6% of the height of monoisotopic peak.
Figure 1. The IROA peaks. Molecule shown is the 6-carbon molecule arginine. Green: Arginine C12 envelope peaks labeled with U-5% 13C. Blue: Arginine C13 envelope peaks labeled with U-95% 13C.