Method development for mass spectrometry based molecular characterization of fossil fuels and biological samples

In an analytical (chemical) method development process, the sample preparation step usually determines the throughput and overall success of the analysis. Both targeted and non-targeted methods were developed for the mass spectrometry (MS) based analyses of fossil fuels (coal) and lipidomic analyses of a unique micro-organism, Gemmata obscuriglobus. In the non-targeted coal analysis using GC-MS, a microwave-assisted pressurized sample extraction method was compared with the traditional extraction method, such as Soxhlet. On the other hand, methods were developed to establish a comprehensive lipidomic profile and to confirm the presence of endotoxins (a.k.a. lipopolysaccharides, LPS) in Gemmata..;The performance of pressurized heating techniques employing hot-air oven and microwave irradiation were compared with that of Soxhlet method in terms of percentage extraction efficiency and extracted analyte profiles (via GC-MS). Sub-bituminous (Powder River Range, Wyoming, USA) and bituminous (Fruitland formation, Colorado, USA) coal samples were tested. Overall 30-40% higher extraction efficiencies (by weight) were obtained with a 4 hour hot-air oven and a 20 min microwave-heating extraction in a pressurized container when compared to a 72 hour Soxhlet extraction. The pressurized methods are 25 times more economic in terms of solvent/sample amount used and are 216 times faster in term of time invested for the extraction process. Additionally, same sets of compounds were identified by GC-MS for all the extraction methods used: n-alkanes and diterpanes in the sub-bituminous sample, and n-alkanes and alkyl aromatic compounds in the bituminous coal sample.;G. obscuriglobus, a nucleated bacterium, is a micro-organism of high significances from evolutionary, cell and environmental biology standpoints. Although lipidomics is an essential tool in microbiological systematics and chemotaxonomy, complete lipid profile of this bacterium is still lacking. In addition, the presence of LPS and thus outer membrane (OM) in Gemmata is unknown.;Global lipidomic analysis of G. obscuriglobus showed fatty acids (FAs) in the range C14 - C22, with octadecanoic and cis-9 hexadecenoic acids (C18:0 and &ohgr;c9 C16:1) being the two most abundant FAs. Thirteen different Gram-negative specific 3-hydroxy fatty acids (3-HOFAs) and eukaryote specific sterols (C30; four in number) were identified. Additionally, like a eukaryotic cell, a polyunsaturated fatty acid (PUFA; tent. &ohgr;3 C27:3) has also been discovered.;The targeted lipidomic study found a series of novel biomarkers in G. obscuriglobus. Compositional analysis of LPS confirmed eight different 3-HOFAs and a sugar-acid, 2-keto 3-deoxy-D-manno -octulosonic acid (Kdo). These two groups of compounds, being unique to a Gram-negative LPS, confirmed the presence of OM in G. obscuriglobus. Moreover, compositional analyses by GC-MS also confirmed glucosamine and hexose and heptose sugars in the LPS. These compositional information obtained from GC-MS analyses were combined with molecular/structural information collected from Matrix-assisted laser desorption and ionization-time of flight (MALDI-TOF) MS. The MALDI-TOF MS showed a cluster of ions separated by 14 u, from m/z 2017.16 to 2143.28. For the most intense ion at m/z 2087.22, a tentative hexa-acylated lipid A structure has been proposed. Identifications of multiple 3-HOFAs by GC-MS and a cluster of ions in MALDI suggest presence of multiple lipid A species, i.e., heterogeneous lipid A molecule, in G. obscuriglobus..