Determination of ubiquinone and related metabolites in zebrafish embryos by LC-HRMS.

Introduction Characterization of lipidome is an emerging topic in metabolomics and the contribution of HPLC-HRMS could be an important tool in the elucidation of biochemical pathways. One goal of this work is to provide a rapid, selective and sensitive method to measure ubiquinone and related lipids concentration in zebrafish embryos and to apply it to metabolism studies. Subsequently untargeted analysis of related lipophilic compounds becomes achievable. Ubiquinone (also known as coenzyme Q10) plays an essential role in the mitochondria electron-transport chain. It is a interesting molecule shown to play an important antioxidant role in the cardiovascular system and it is the only endogenously synthesized lipid-soluble antioxidant. Zebrafish (Danio rerio) is an established model for studying toxicology and understanding human diseases. Methods Embryos and adult fishes were raised and maintained under standard laboratory conditions. Biological samples were extracted by LLE with various organic solvents. HPLC-HRMS analyses were accomplished on a Dionex Ultimate 3000 LC system coupled with a LTQ-Orbitrap instrument, with ESI and APCI interfaces. C4, C8 and C18 RP columns were tested for separation. Here we investigate the ionization modes, the ex vivo analytical sensitivity and the fragmentation mechanisms of ubiquinone and related compounds. The developed methodology will be applied to study samples of zebrafish embryos. Preliminary data In the first steps of method development we optimized the chromatographic separation of highly lipophilic ubiquinone related compounds on RP-HPLC columns and studied the sensitivity of different ionization modes (positive vs. negative ion mode, APCI vs. ESI, proton vs ammonium vs. sodium vs. lithium adducts formation). A fragmentation study of the analytes was also done evidencing a double neutral loss of formaldehyde as major pathway. A full validation study was then completed to make possible quantitative determination on biological samples. Accuracy, precision, LLOQ, linearity, extraction recovery were evaluated. Then to confirm the high similarity between zebrafish and higher vertebrates at the cellular and physiological levels we determined the variation of concentration of coenzyme Q10 at various zebrafish growth steps and compared it with similar metabolites as coenzymes with shorter isoprene chain (Q9 to Q6). Finally a study to characterize the untargeted lipidome of embryo samples is ongoing to resolve the complex mixture of compounds giving mono-charged ions in the range 700-1000 m/z. Novel aspects Rapid quantification of ubiquinone and untargeted lipidomic analysis in ex vivo zebrafish samples for biochemical studies.