Metabolite identification in early stage discovery is generally performed prior to the availability of a radiolabel. In these cases, the detection and identification of xenobiotic metabolites is often aided by the use of either data-dependent or post-acquisition filtering methods including mass defect filtering (MDF), sample/control comparison, and screening for predicted metabolites. These methods have been employed to some degree on each of the systems described above, and we will present the results we have obtained employing these methodologies on each instrument in the identification of xenobiotic metabolites generated in both plant and soil systems. Of particular focus is the application of ultra high resolution to separate and detect the isotopic fine structure of metabolites containing natural sulfur (32S/34S), oxygen (16O/18O) isotopes, as well as 13C isotope labels. We will present several examples of the use of this information in environmental metabolite identification at trace levels. In regulatory metabolite ID, radiolabeled metabolites are typically profiled using conventional LC to allow the injection of sufficient material for online radiochemical detection. These metabolite samples, which may take weeks-months to form, and must be identified rapidly to avoid jeopardizing critical registration deadlines. To utilize these precious samples most efficiently, the post-column effluent is generally split between the MS and a RAM (Yt:Si cell) and the effluent from the RAM cell is collected into a 96-well plate. This allows the recovery of most of the LC-fractionated sample. If additional spectra are required for identification, these well contents can then be assayed to localize the peaks of interest, and then analyzed using 2-D (trap and elute) nanoLC combined with ultra high resolution MS, MS/MS, and MSn using the Thermo Q-O-LIT instrument. These methods may provide MS signal enhancement (100-1000 fold) via on-column concentrations coupled to nano-separations, as well as the detection of isotopic fine structure. We will discuss the application of each of these techniques to the identification of trace level metabolites produced in whole plant, plant cell culture, and soil metabolism for several agricultural chemicals.

Identification of environmental metabolites using combined high resolution UPLC-QqTOF and ultra high resolution NanoLC-Q-Orbi-LIT based approaches

248th National Meeting (2014)

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