Identification & characterization of polar metabolites derived from pharmaceuticals and agrochemicals present in complex biological matrices can be challenging & time-consuming. Most of these polar metabolites are not amenable to LC/MS analyses & often do not possess a chromophore to LC/UV analyses. Hence, a number of analytical techniques or chemistry effort are often required to unambiguously assign chemical structures of such metabolites. A case study is presented herein which required a combination of chromatographic and analytical techniques as well as strategic chemical synthesis/derivatization to elucidate the structure of a very polar metabolite derived from a 14C-FMC herbicide. A distinct polar radioactive peak was observed in radio-chromatograms of in vivo sample extracts of this compound. In vivo studies were performed in male & female SD rats administered a low single oral dose (200 mCi/kg) of the 14C-FMC herbicide. This polar radioactive peak was observed to elute at a retention time of ~3 min on a HPLC column and was not amenable to mass spectral analyses. To facilitate the identification of this radioactive peak, a study was conducted whereby urine was collected from male rats administered a high single oral dose of non-radioactive FMC herbicide and spiked with incurred radioactive urine from low dose study. The urine was loaded onto a C18 cartridge and the polar peak separated from other metabolites and endogenous components. The fraction containing the radioactive polar peak was lyophilized and derivatization was attempted with triphenylphosphine, dipyridyldisulfide and hydrazinoquinoline at 60°C for 3 days. The derivatized polar peak was further purified using a fraction collector coupled to a HPLC system. The collected fraction was then concentrated for LC/MS and NMR analysis. A single radioactive component was obtained after purification of the polar derivatized peak. The molecular ion, (M+H)+ at m/z 203, and the characteristic fragmentation pattern of the hydrazinoquinoline derivative suggested that the polar metabolite was carbamic acid. The elemental composition of this derivative was further confirmed by accurate mass data obtained on an Orbitrap high resolution MS. The results demonstrate that a combination of optimum chromatographic separation and LC/MS derivatization technique provides a viable approach to identify low molecular mass polar metabolites.

Identification and characterization of a polar metabolite produced from a FMC herbicide administered to Sprague-Dawley rats

254th National Meeting (2017)

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