"Finding the needle in the haystack"

January 05, 2017

"Finding the needle in the haystack:" Using the unique or unusual isotope pattern of radiolabeled compounds to find low level metabolites in complex matrices.

Most environmental metabolite ID work involves the use of radiolabeled compounds and radiochromatography to trace and quantify a compound and its metabolites. Frequently, a compound assumed to be radiolabeled at only one atom turns out to be a mixture of radiolabels with a unique isotope pattern or isotopic fingerprint. This situation can be especially useful in finding low level metabolites.

The current regulatory level only requires identification of metabolites above 5 to 10% of the applied radioactivity, however, there are situations where multiple metabolites with similar chromatographic properties co-elute and can appear as one component, thus triggering the need for further testing and resulting in increased cost for product registration. This situation can be avoided by conducting additional LC/MS analyses to find and then identify the individual low level co-eluting compounds appearing to be a single component on the radiochromatogram. Finding/identifying the multiple components can show, for example, that a peak that appears to have one component with 10% of the AR is really a peak composed of 3 components with 3%, 3%, and 4% of the AR; none of which would require further work.

Filtering techniques in LC/MS can help identify low level metabolites into the range of 1-2% of the applied radioactivity if the compound or labeled compound provides a traceable fingerprint. The analyst can filter either a spectrum or a chromatogram for a given isotope pattern or isotopic fingerprint with a specified tolerance. The filtered spectrum draws the analyst’s attention to the ions that are compound-related. With the use of specialized software, the ions in the spectrum matching the fingerprint can be color-coded, allowing rapid removal of the ions which do not match the fingerprint.

Detection of low level metabolites can also be helpful in describing the degradation pathway for compounds assumed to be persistent or for slowly degrading compounds. In addition to radiolabeled compounds, this filtering process can also be applied when there are chlorines, bromines or other atoms with a strong natural isotopic fingerprint in the molecule.