Regulatory studies for metabolism in plant and animal matrices commonly use agrochemicals with 14C radiolabeled test substances. At the initiation of a study, isotopic dilution of the radiolabeled test material determines the radiochemical specific activity of the test substance applied to the test system, which affects the limits of quantification of radioisotope detection. In conjunction with the specific activity, the 14C labeled position affects the ability to distinguish metabolites originating from the test material in mass spectral (MS) data. The location of the 14C tracer can be exploited to aid identification of metabolites via liquid chromatography-mass spectrometry analysis (LC-MS) by adjusting the isotopic enrichment to give unique 12C and 14C isotope ratios. Uniform ring labeled compounds may be necessary to trace test substance metabolites, but generally do not give the advantage of a unique isotopic ratio with high intensity. Dilution of the isotopic enrichment is mainly due to the mixture of incorporated radiolabel (14C1, 14C2, 14C3, 14C4, 14C5, 14C6) resulting in spectra that are not as easily differentiated from other endogenous materials without extensive purification. The presence of halogens in the tests substance and its products may also contribute to the observed MS isotopic ratio due to naturally occurring heavy isotopes, but during biotransformation any halogens present may be lost. Therefore, a strategy of incorporating stable labeled analogs such as 13C should be considered. Examples where use of a single carbon incorporation compared to uniform ring labeled incorporation of the 14C isotope will be presented. The molecular ion isotopic patterns observed by LC-MS analysis will be compared, along with examples of how halogen presence affects the isotopic ratios. The incorporation of 13C stable labeled analogs to control the predicted unique isotopic ratios of test substances and metabolic products will be presented as a strategy to allow for easier metabolite identification in less purified matrices.

Considerations for selection of 14C radioactive tracers and 13C stable label analogs to aid metabolite identification by mass spectrometry

256th National Meeting (2018)

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