National Research Council of Canada, Institute for Marine Biosciences, 1411 Oxford St., Halifax, Nova Scotia, B3H 3Z1, Canada.
Rapid Commun Mass Spectrom. 2012 Feb 15;26(3):345-54. doi: 10.1002/rcm.5336.
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.
已经开发出一种使用液相色谱-高分辨质谱联用（LC/HRMS）来鉴定海洋生物毒素 13-去甲基螺旋内酯 C 代谢物的策略。通过与人类肝微粒体孵育在体外生成代谢物。通过选择已知含有环亚胺环的螺旋内酯毒素的特征性共同片段离子的前体离子，建立了代谢物列表。随后使用精确质量测量来确认每个生物转化产物的分子式。使用这种方法，总共成功鉴定了 9 种 I 相代谢物，其质量精度偏差小于 2 ppm。观察到的生物转化包括羟化、二羟化、季甲基氧化为羟甲基或羧酸基团、脱氢和羟化以及去甲基化和二羟化反应。在第二步中，进行串联质谱（MS/MS）以阐明代谢物的结构。利用谱中的独特片段离子，确定了三种主要代谢物 13,19-二去甲基-19-羧酸螺旋内酯 C、13,19-二去甲基-19-羟甲基螺旋内酯 C 和 13-去甲基-17-羟基螺旋内酯 C 的结构。在与人类肝微粒体孵育 32 小时的选定时间点监测 13-去甲基螺旋内酯 C 及其代谢物的水平。确定在孵育 2 小时后，13,19-二去甲基-19-羧酸螺旋内酯 C 成为主要代谢物。13-去甲基螺旋内酯 C 的稳定性图显示其代谢呈一级动力学，内在清除率计算为 41 μL/min/mg，表明首过代谢可能有助于限制 13-去甲基螺旋内酯 C 的口服毒性。
