Acidic methanolysis v. alkaline saponification in gas chromatographic characterization of mycobacteria: differentiation between Mycobacterium avium-intracellulare and Mycobacterium gastri

Abstract

Mycobacterium avium-intracellulare and M.gastri were analyzed with capillary gas chromatography after each strain had been subjected to acidic methanolysis or to alkaline saponification followed by methylation. Prominent peaks of myristic, palmitoleic, palmitic, oleic, stearic and tuberculostearic acids were found in the chromatograms of both species, whereas 2-octadecanol and 2-eicosanol were detected only in M. avium-intracellulare. In initial runs, both of the derivatization principles yielded virtually identical chromatograms for a given strain. After repeated injections of extracts from alkaline saponification, however, the alcohol peaks showed pronounced tailing and finally almost disappeared from the chromatograms. This disadvantage, which was not observed when only acid methanolysis was used, could be overcome with trifluoroacetylation. Restored peak shape of the underivatized alcohols could be achieved by washing the cross-linked stationary phase in the capillary tubing with organic solvents. The study demonstrated the importance of conditions which enable separation of 2-octadecanol and 2-eicosanol when gas chromatography is used for species identification of mycobacteria.