The objective of the current research project relies on implementation of solvent-free, green and environmentally friendly solid phase microextraction (SPME) sample preparation alternative in the area of complex sample characterization. The advantages that the technique offers in comparison to traditional methods of sample preparation including solvent-free implementation, short sample preparation times, small sample amount requirements, advanced automation capability and minimization of matrix effects are effectively employed during ex vivo and laboratory investigations of complex samples. More important, the underlying features of the technique including miniaturized format, nonexhaustive extraction recoveries and on-site compatibility were fully exploited in order to investigate the metabolome of biological systems directly on the site. Hence, in vivo SPME extraction format was employed in direct immersion SPME sampling of biological systems, hence eliminating the crucial prerequisites associated with multiple preparative steps and incorporation of metabolism quenching that are encountered during implementation of traditional sample preparation methods in global metabolite analysis. Furthermore, in vivo sampling format was hyphenated to comprehensive two-dimensional gas chromatography – time-of-flight mass spectrometry (GCxGC-ToFMS) for high-resolution sampling of volatile and semivolatile metabolites in ‘Honeycrisp’ apples.
The initial stages of the project involved evaluation of performance characteristics of commercial SPME extraction coatings in terms of extraction selectivity, extraction sensitivity and desorption efficiency by employing headspace SPME analysis of both aqueous standards spiked with representative volatile and semivolatile metabolites as well as the apple homogenate. DVB/CAR/PDMS coating was selected on the basis of optimum metabolite coverage and extraction sensitivity and was consequently employed during ex vivo and in vivo sampling assays corresponding to determination of volatile and semivolatile metabolites. The former extraction methodology incorporated appropriate sample preparation steps for quenching metabolic activity so that the relevant metabolome profile is not biased against unstable metabolites and those that are susceptible to inter-metabolite conversions which adversely impact preservation of metabolite identity. The two sample preparation assays were compared in terms of metabolite coverage and analytical precision in order to identify SPME route toward characterization of more representative metabolome and determination of instantaneous and more ‘true’ metabolism snapshoot. This is the first report illustrating the implementation of in vivo direct immersion SPME assay for non invasive determination of endogenous fruit metabolites whose profiles and contents are highly correlated to a multitude of influential fruit quality traits.
| Identifer | oai:union.ndltd.org:WATERLOO/oai:uwspace.uwaterloo.ca:10012/7006 |
| Date | January 2012 |
| Creators | Risticevic, Sanja |
| Source Sets | University of Waterloo Electronic Theses Repository |
| Language | English |
| Detected Language | English |
| Type | Thesis or Dissertation |
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