Strategic identification of in vitro metabolites of 13-desmethyl spirolide C using liquid chromatography/high-resolution mass spectrometry.
A strategy to identify metabolites of a marine biotoxin, 13-desmethyl spirolide C, has been developed using liquid chromatography coupled to high-resolution mass spectrometry (LC/HRMS). Metabolites were generated in vitro through incubation with human liver microsomes. A list of metabolites was established by selecting precursor ions of a common fragment ion characteristic of the spirolide toxin which was known to contain a cyclic imine ring. Accurate mass measurements were subsequently used to confirm the molecular formula of each biotransformation product. Using this approach, a total of nine phase I metabolites was successfully identified with deviations of mass accuracy less than 2 ppm. The biotransformations observed included hydroxylation, dihydroxylation, oxidation of a quaternary methyl group to hydroxymethyl or carboxylic acid groups, dehydrogenation and hydroxylation, as well as demethylation and dihydroxylation reactions. In a second step, tandem mass spectrometry (MS/MS) was performed to elucidate structures of the metabolites. Using the unique fragment ions in the spectra, the structures of the three major metabolites, 13,19-didesmethyl-19-carboxy spirolide C, 13,19-didesmethyl-19-hydroxymethyl spirolide C and 13-desmethyl-17-hydroxy spirolide C, were assigned. Levels of 13-desmethyl spirolide C and its metabolites were monitored at selected time points over a 32-h incubation period with human liver microsomes. It was determined that 13,19-didesmethyl-19-carboxy spirolide C became the predominant metabolite after 2 h of incubation. The stability plot of 13-desmethyl spirolide C showed first-order kinetics for its metabolism and the intrinsic clearance was calculated to be 41 μL/min/mg, suggesting first-pass metabolism may contribute to limiting oral toxicity of 13-desmethyl spirolide C.
National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford St., Halifax, Nova Scotia, B3H 3Z1, Canada.
Joseph P M
JP
Hui
J
J
Stuart Grossert
Murray J
MJ
Cutler
Jeremy E
JE
Melanson
D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D006900:Hydroxylation; D007700:Kinetics; D008387:Marine Toxins / Q000737:chemistry / Q000378:metabolism*; D008862:Microsomes, Liver / Q000378:metabolism; D010084:Oxidation-Reduction; D013141:Spiro Compounds / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
C517170:13-desmethylspirolide C; D008387:Marine Toxins; D013141:Spiro Compounds