The unique IROA labeling pattern ensures that the monoisotopic peaks and the
carbon envelope of the associated isotopic peaks (M-1 etc.) can be detected during LC-MS. The carbon envelope differentiates the IROA-IS from natural abundance peaks (and is used to identify compounds of interest and exclude artifacts that may look otherwise similar.
The IROA-IS is a true Internal Standard and can be spiked into any natural abundance experimental sample (cells, tissue biopsy, plant material, blood, etc.) and all the IS peaks may be easily identified using the ClusterFinder software according to the presence of their characteristic M-1 peak and associated carbon envelope. It provides enough information for complete identification and quantitation of samples without the need for chromatographic base-line correction.
Fundamental to the IROA concepts (and inherent in the name Isotopic Ratio Outlier Analysis) is the fact that the ratio of the C-12 envelope to the C-13 envelope is unaffected by suppression even though both the C-12 and C-13 isotopomeric sets may be strongly suppressed. This has afforded a mechanism for suppression correction that has been built into ClusterFinder.
The algorithm for suppression correction starts by determining the true ratio of the total number of molecules in their respective C12 and C13 envelopes and then multiplying this ratio with the “unsuppressed value” of the molecule at hand.
-
- The “least unsuppressed value” can be the largest C13 value seen within an experiment for each compound. This value can then be used within an experiment and will recover the effects of suppression as seen in the experiment.
- The “least unsuppressed value” may be the largest C13 value seen in a sample that is otherwise a blank (i.e., containing no sample).
- A more accurate, and enduring value for the “unsuppressed value” can be determined in a specific experiment in which the internal solution is serially diluted and analyzed in the absence of any other sample.
- If there is a quantitatively accurate determination of the concentration of the internal standard for the peak in question, then the ratio may simply be multiplied by this concentration to obtain an approximately accurate concentration value.
It should be noted that options 1 and 2 require no further data to be generated external to the experiment, while options 3 and 4 rely on experimentally established quantitative values, that once established may be used at any time if an identical aliquot size of the same internal standard is used to resolvate the dried sample. Options c and d represent solutions that can be repeatedly used for longer periods of time, over many experiments, or even for very large studies using hundreds or thousands of samples.
