Development of GCxGC/TOF-MS metabolomics for use in ecotoxicological studies with invertebrates

Abstract

The majority of metabolomic studies used in ecotoxicology have implemented 1H NMR analysis. Despite constant improvement, major limitations of NMR-based techniques include relatively low sensitivity that results in an examination of a limited number of metabolites. An alternative approach is the use of liquid or gas chromatography (GC) for separation of metabolites and mass spectrometry (MS) for their quantification and identification. The objective of our study was to develop a two dimensional GC coupled with time of flight MS (GCxGC/TOF-MS) coupled with multivariate analysis to compare metabolite profiles of Diporeia under different environmental conditions. We compared metabolite profiles between Diporeia collected from Lake Michigan (declining populations) to those residing in Lake Superior (stable populations), and also between Diporeia exposed to a chemical stressor (atrazine) and controls. Overall, 76 and 302 total metabolites were detected from the lake comparison and atrazine studies, respectively. Many of the identified metabolites included fatty acids, amino acids, and hydrocarbons. Furthermore, we observed unique and almost non-overlapping metabolite profiles in both studies. In conclusion, we established the feasibility of using GCxGC/TOF-MS for detecting metabolites as well as developed software to align and merge chromatographic peaks to compare metabolite differences between invertebrate groups sampled under different environmental conditions. This ability to detect unique metabolite profiles under different environmental conditions will increase our undertsanding on the physiological processes and whole-organism reponses occuring as a result of exposure to different environmental stressors.