James Gilbart

Modifications in the alditol acetate method for analysis of muramic acid and other neutral and amino sugars by capillary gas chromatography-mass spectrometry with selected ion monitoring.

Two alditol acetate methods for the gas chromatographic (GC) analysis of neutral and amino sugars were compared. Following sodium borohydride reduction, one method uses methylimidazole as an acetylation catalyst without prior removal of water or borate salts and the other method uses sodium acetate after removal of borate and water. Depending on the acetylation conditions, muramic acid produced different derivatives. With methylimidazole, reliable derivatization of muramic acid was not possible, although other sugars derivatized reliably. With sodium acetate, all sugars tested were reproducibly derivatized. The utility of the sodium acetate method is shown by the trace GC-mass spectrometric analysis of muramic acid and rhamnose derived from bacterial peptidoglycan-polysaccharide complexes in mammalian tissue.

Rhamnose and muramic acid: chemical markers for bacterial cell walls in mammalian tissues

Abstract An improved analytical chemical method for the quantitation of low levels of streptococcal cell walls in inflamed mammalian tissues was developed. Rhamnose (a component of the group-specific polysaccharide) and muramic acid (a component of the peptidolglycan) of group A streptococci were detected in experimental tissues at levels as low as 1 ng/mg using a modification of the alditol acetate procedure and selected ion monitoring gas chromatography-mass spectrometry. Rhamnose and muramic acid were not detected at these levels in normal mammalian tissues and are thus excellent chemical markers for bacterial debris.

Carbohydrate profiling of bacteria by gas chromatography-mass spectrometry: Chemical derivatization and analytical pyrolysis

Carbohydrate profiling by gas chromatography-mass spectrometry is a powerful tool for the identification and detection of bacteria. Its increasing applicability in the microbiology laboratory is illustrated by three examples. In the first, differentiation of legionellae by their sugar composition was determined with alditol acetate derivatization followed by selectedion monitoring. In the second example, a carbohydrate pyrolysis product fromStreptococcus agalactiaewas used to differentiate group B streptococci from other Lancefield groups after direct sampling from culture plates. The third example employed the carbohydrates rhamnose and muramic acid as chemical markers for the direct detection of bacterial cell wall degradation products in mammalian tissues. The analysis of carbohydrate markers for bacteria by gas chromatography-mass spectrometry has great potential for use in clinical identification of isolated bacteria as well as in the rapid diagnosis of bacterial infections without prior culture.

Profiling, structural characterization, and trace detection of chemical markers for microorganisms by gas chromatography-mass spectrometry☆

Abstract Gas chromatography-mass spectrometry (GC-MS) is an important technique for the identification and detection of bacteria and bacterial constituents. Certain classes of structural components, or secreted metabolites, are useful as chemical markers for different groups of organisms. GC-MS methodologies suitable for applications in analytical microbiology are summarized. In some instances, flame ionization GC is adequate for chemotaxonomic analysis of bacterial cells; MS, however, offers increased selectivity and sensitivity and allows positive identification of components. Trace detection of chemical markers in biological fluids or other complex matrices in most instances requires careful sample cleanup combined with selected ion monitoring.

Analysis of the amino acid and sugar composition of streptococcal cell walls by gas chromatography-mass spectrometry.

A procedure for determining the amino acid and sugar composition of streptococcal peptidoglycan-polysaccharide complexes by capillary gas chromatography-mass spectrometry (GC-MS) was established. Amino acids are analysed as butyl heptafluorobutyl derivatives and sugars as alditol acetates. These two different groups of compounds are derivatized independently but chromatography in both cases utilizes the same OV-1701 fused-silica capillary column which simplifies GC-MS analysis. The butyl heptafluorobutyl procedure incorporates new pre- and post-derivatization clean-up steps. Additionally, selected-ion monitoring MS allows amino acids to be readily analysed without interference from background noise.

Analytical Microbiology: A Perspective

The term “Analytical Microbiology” describes the application of analytical chemistry to identification, structure elucidation, systematics, diagnosis, and detection in microbiology. The most widely applied instrumental chemical techniques have been the various forms of gas chromatography (GC) and mass spectrometry (MS). These and other analytical techniques (e.g., high performance liquid chromatography, HPLC) and the “hyphenated” methods (including GG-MS, HPLC-MS and MS-MS) can be used to detect trace levels and to identify monomers, oligomers, or polymers derived from microorganisms. Chemical information from these analyses can be of great benefit in medicine, ecology, biotechnology, as well as in the food and pharmaceutical industries. Classical microbiological or biochemical procedures may not be sufficiently sensitive, selective, quantitative, or rapid in some instances. Instrumental approaches, hitherto considered the realm of the chemist, have found application in microbiological analysis. We perceive four major areas in analytical microbiology where chromatography and mass spectrometry are having impact.

Profiling and Detection of Bacterial Carbohydrates

Carbohydrates are a major class of structural components in bacterial cell envelopes. Sugar profiles can differentiate and identify isolated bacteria. Carbohydrates can also serve as chemical markers for direct detection of bacteria in complex matrices such as mammalian body fluids and tissues. This chapter reviews the composition of some bacterial polysaccharides and their characteristic sugars. Preparation and analysis of derivatives of these sugar monomers by gas chromatography (GC) and mass spectrometry (MS) is described. A modified alditol acetate procedure developed in our laboratories is presented along with some examples of its use.