Identifying Isotope Patterns in Complicated Mixtures

In drug discovery and development, metabolite identification unveils the fate of xenobiotic compounds and contributes to comprehension of their physiological, pharmacological, and toxicological potential. Radiolabelled compounds are highly valued in these studies for the purpose of comparing mass balances, biotransformation pathways, and in vitro efflux transport studies.

Labeled metabolites can be efficiently separated by chromatography, and radio detection is both sensitive and selective, but to derive insight into the structure of the metabolite a more involved approach using LC/MS is generally used. LC/MS is rapid and sensitive, however, low abundance metabolites can be hidden in datasets containing significant endogenous background and noise.

The presence of a significant radioisotope can alter the isotope pattern significantly enough that labeled compounds become sufficiently distinctive to find with tools that are able to identify isotope patterns in MS data.

One such tool is a recently developed filter for isotope abundances that can be applied to find components with characteristic isotope patterns in the full scan mass spectra of LC-MS datasets. The ability to identify non-natural isotope patterns of radiolabeled metabolites should make it simpler to distinguish the components of interest in the types of samples referred to above.

A prototype of our Special Isotope Cluster filter was implemented in the ACD/IntelliXtract software and tested for its ability to recognize labeled metabolites in an ion trap dataset of ¹⁴C-buspirone incubated with human liver microsomes, which had been analyzed previously.

Compared to the normal isotope ratio of buspirone, the peaks in the spectrum show that the A+2 peak or '12C+2' is enhanced. One may calculate a theoretical isotope pattern or, as in this case, one may examine the experimental data to determine the ratio. After one identifies the components and potentially annotates possible metabolites using IntelliXtract, the isotope ratios in the Special Isotope filter can be specified and the results in the Table of Mass Chromatograms can be filtered for those components with an isotope ratio in their spectrum that match the expected ratio, i.e., a Pass value annotated for that particular isotope.

The same algorithm used for the Special Isotope Cluster filter can also be applied for extracting chlorinated, brominated, or sulfinated compounds based on their characteristic isotope patterns.