Multi-dimensional, comprehensive sample extraction combined with LC-GC/MS analysis for complex biological samples: application in the metabolomics study of acute pancreatitis

Abstract

An analytical strategy employing multi-dimensional sample extraction together with optimal analytical platforms was established. The proposed workflow was based on the fact that most biological samples are complex in terms of molecular species as well as their properties, e.g. hydrophobicity. A few new methods based on methyl tert-butyl ether (MTBE) extraction, which was developed for lipid extraction and used perform a lipidomic study on the upper layer fraction of the MTBE extraction system and a metabolomics analysis by mixing the upper and lower fractions from the same sample, have been developed to acquire more information from samples, and in the present work we established a similar method employing the MTBE procedure to separate samples into polar and apolar fractions, then associated each with an adapted subsequent analysis technique. The polar fraction was silylanized for GC/MS analysis, and apolar lipids were analyzed by reverse phase liquid chromatography (RPLC) on a T3 column. The proposed strategy was applied to investigate acute human pancreatitis and was compared with results obtained by conventional LC/MS-only and GC/MS-only analysis. Features obtained in our LC-GC/MS data outnumbered either the LC/MS-only or GC/MS-only data. Furthermore, with the aid of multivariate analysis, differential metabolites from pancreatitis vs. normal pancreas, mainly amino acids and phospholipids, were identified. This work demonstrates that the proposed analytical strategy is a promising tool to perform metabolomics. In addition, it improved our understanding of the pathogenesis of acute pancreatitis and provided potential biomarkers for AP diagnosis.