Metabolic profiling of biologically-based samples – new profiling capabilities reveal new information
Creators: name: Kouremenos, Konstantinos
Publisher: name: RMIT University
Year: 2024

description: In the work underlying this thesis, comprehensive two-dimensional gas chromatographic (GCxGC) methods have been developed which can be used to compare metabolic profiles from both human and plant metabolic samples. GCxGC was used to extend the GC approach in this area by using 2DGC methods, which provide greater separation power, lower detection limits (greater sensitivity, and therefore cleaner MS spectra), smaller sample amounts and less overall analysis times. <br><br>A reliable microwave derivatization protocol for metabolomic analysis has been developed through the analysis of a set of standards which included a wide range of metabolites of interest to metabolic profiling. The method presented is rapid (significant decrease in derivatization time, therefore overall decreasing the total analysis time), sensitive and produces less side reactions (a more complete derivatization reaction). It has been successfully applied to two main application areas: human (urine) and plant (ginseng) metabolite profiling.<br><br>In the infant urine metabolite profiling of organic acids for the diagnosis of organic acidurias, abnormal profiles were obtained from five ‘diseased’ patient samples and compared to five ‘normal’ patient samples. GCxGC with ToFMS detection was useful in separating isomeric organic acids that were otherwise not resolved using 1DGC. Crotonyl glycine was also discovered in the mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase sample as a possible biomarker for the specific disorder. Quantification was investigated using stable isotope dilution analyses of glutaric, glyceric, orotic, 4-hydroxybutyric acids and 3-methylcrotonylglycine. Correlation coefficients for linear calibrations of the analytes ranged from 0.9805 to 0.9993 (R<sup>2</sup>) and analytical recoveries from 77 and 99%. <br><br>The work undertaken in the plant metabolic profiling area comprised of the comparison of four, five and six year aged <i>panax ginseng </i>samples through the detection, identification and quantification of volatiles using HS-SPME-GCxGC/TOFMS, and full-metabolite screens for the study of primary and secondary active metabolites using GC×GC/ToFMS. In the SPME study, major compounds identified in the samples analyzed were sesquiterpene hydrocarbons and sesquiterpene alcohols, with most of the previously known active compounds present in the five followed by the six year aged samples (examples include α-panasinene, ß-panasinene, α-humulene and α-neoclovene). In the full-metabolite screen, primary active compounds identified and quantified included α-galactopyranosyl-galactopyranose (a trisaccharide) and protopanaxadiol and protopanaxatriol (two glycosides); these primary active compounds were only found in the five and six year aged samples. Secondary active compounds included monosaccharide and disaccharide sugars. Quantification was performed using a 10 metabolite standard mixture with concentration levels of standards ranging from 0.01 to 100 µg/ml. R<sup>2</sup> values obtained were mainly higher than 0.980, with RSD (%) usually less than 5%. Concentration levels found for the primary active compounds ranged from 0.07 - 0.17 µg/ml for protopanaxadiol, with protopanaxatriol concentrations ranging from 0.10 - 0.15 µg/ml in the six year aged samples.<br>