Trace detection and identification of bacterial constituents including D-alanine, D-glutamic acid, and glucitol phosphate by negative chemical ionization mass spectrometry and analytical pyrolysis

Bacteria contain unique components useful as chemical markers for classification or trace detection. Gas chromatography-mass spectrometry (GC-MS) can detect and identify extremely low concentrations of compounds but volatile components are required. This dissertation investigates two different ways to produce such volatile compounds from the macromolecular structure of microorganisms--analytical pyrolysis and chemical derivatization. In the introductory chapter, relevant principles of GC and MS were reviewed.; Electron impact and chemical ionization MS was employed following pyrolysis GC to elucidate the structural origin of a chemical marker for group B streptococci. The chemical marker was identified as a dehydration product of glucitol-6-phosphate. General approaches to microbial analysis by analytical pyrolysis might be based on a closer examination of pyrolysates of carbohydrate origin.; GC-MS using selected ion monitoring with negative ion detection and methane chemical ionization was employed to quantitate D-alanine, a marker for bacterial peptidoglycan, in mammalian tissues. D-alanine from bacterial peptidoglycan was obscured by D-alanine from racemized protein L-alanine. To overcome this problem, samples were enzymatically treated and hydrolyzed in deuterated hydrochloric acid. D-alanine derived from protein was labelled with deuterium and bacterial D-alanine remained unlabelled, enabling differentiation. Butyl heptafluorobutyryl derivatives of the D- and L-amino acids were separated on a fused silica capillary column coated with Chirasil-L-Val.; The determination of D-/L-alanine and D-/L-glutamic acid ratios in whole microbial cells by GC-MS was evaluated. Electron impact and negative ion chemical ionization methods were compared; both approaches provided excellent results. Samples were hydrolyzed in deuterated hydrochloric acid and converted to butyl heptafluorobutyryl derivatives. The efficacy of two different pre-derivatization clean-up steps (disposable C{dollar}sb{lcub}18{rcub}{dollar} versus ion exchange columns) were compared; C{dollar}sb{lcub}18{rcub}{dollar} columns produce equivalent results to ion exchange, but are easier to use. MS analysis revealed a shift of two mass units in the base peak of D-glutamic acid, clarifying previous reports of irreproducibility and lack of sensitivity, and permitting the use of D-glutamic acid as a chemical marker.