Development and evaluation of sample preparation procedure for human plasma samples in LC/MS-based metabolomics

University essay from Uppsala universitet/Avdelningen för analytisk farmaceutisk kemi


The aim of this master thesis project was to develop methods for sample preparation and analysis by LC-MS suitable for global metabolomics of human plasma samples. In this thesis six different methods was tested, based on precipitation, solid phase extraction (SPE), and ultrafiltration. The methods differ in their mechanisms of action, but the end goal is the same, to remove the proteins and other high molecular weight compounds from the samples and to retain as many metabolites as possible in a reproducible manner. The LC-MS analysis were performed on a C18 and a HILIC type column using electrospray ionization (ESI) with both positive and negative ionization to cover as much of the metabolome as possible. The MarkerLynx software was then used to extract features from the chromatograms. The coefficient of variation (CV) was calculated and the features with CV > 30 % were removed. The features were then used to compare the methods with each other in order to see whether any of the methods yielded the same capture of features. The largest amount of features was detected for precipitation with MeOH when using a C18 type column. For HILIC type columns precipitation with MeOH with a small addition of H3PO4 resulted in most unique features detected. The ionization mode was found to be less important, compared whit the choice of column, but more features was detected using positive mode than negative mode.

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