A MULTI-DIMENSIONAL METHOD FOR THE ANALYSIS OF HUMAN BLOOD PLASMA METABOLOME

Abstract

<p>The comprehensive analysis of human blood plasma metabolome has been completed using derivatization gas chromatography-mass spectrometry (GC-MS) analysis, liquid chromatography-mass spectrometry (LC-MS) analysis and a comprehensive LC-GC-MS analysis approach wherein LC fractions were collected, derivatized and analyzed using GC-MS. In all cases blood plasma samples were deproteinized using solvent precipitation prior to chromatography and MS analysis.</p> <p>In GC-MS analyses, the progress of all derivatization reactions was monitored by adding 9-anthracenemethanol and 1,3-diphenylacetone to all reaction mixtures; their conversions to 9-anthracenemethanol trimethylsilyl ether and the oxime derivative of 1,3-diphenylacetone were used as measures of the completion of these derivatization reactions. Any reactions with completions less than 99% were repeated.</p> <p>GC-MS analysis of blood plasma samples detected 100 peaks; 44 were positively identified by comparing retention indices and mass spectra with those of authentic standards. LC-MS analyses were conducted on a HILIC column (aminopropyl phase) with MS detection in both negative ion and positive ion modes and resulted in the identification of 97 peaks; 47 were observed in the positive ion mode, 58 in the negative ion mode with 8 peaks observed in both modes.</p> <p>The multi-dimensional LC-GC approach was not designed as a routine analytical method; rather the purpose of this approach was to see how many compounds could be observed in the sample and to obtain better quality mass spectra and retention index values. The LC separation afforded 16 fractions which upon derivatization GC-MS analysis gave an additional 176 peaks from a total of 276 peaks. The MS data from these additional spectra can be used to develop selected ion monitoring GC-MS or tandem mass spectrometry analytical methods. This thesis has demonstrated the power of off-line comprehensive methods to identify compounds that neither the GC nor the LC methods detected.</p>