Determination of molecular species composition of C80 or longer-chain α-mycolic acids in Mycobacterium spp. by gas chromatography-mass spectrometry and mass chromatography

Abstract

The molecular species composition of α-mycolic acids ranging from C68 to C86 in 13 rapidly growing and 12 slowly growing mycobacterial species was determined by gas chromatography, gas chromatography-mass spectrometry, and mass chromatography. In gas chromatographic analysis, the molecular species of α-mycolic acids were well separated as trimethylsilyl ether derivatives of the methyl esters, according to their total carbon numbers. The total carbon and double-bond numbers of mycolic acids at each peak on gas chromatograms were determined from the [M]+, [M - 15]+, and [M - 90]+ ions on the mass spectrum, and straight and branched chain structures were identified by the mass fragment ions [A]+, due to C2-C3 cleavage [R-CH-O-Si(CH3)3]+, and [B]+, due to C3-C4 cleavage [(CH3)3-Si-O-CH-CH(R')-COOCH3]+. The concentration of odd- and even-carbon-numbered mycolic acids, which often overlap each other on gas chromatograms, and the composition of three homologous mycolic acids with different α units (C(22:0), C(24:0), and C(26:0)) were clearly determined by mass chromatography monitoring [M - 15]+ ions and [B - 29]+ ions, respectively. The molecular species composition of α-mycolic acids and their average carbon numbers (av. cn.) as a simple expression of the composition were calculated from the mass chromatograms. Each mycobacterial species examined was demonstrated to possess a characteristic profile of α-mycolic acid composition, and based on this the species were classified approximately into eight groups: (i) C68 to C76 (av. cn. 72), dienoic, possessing a C20 alkyl branch at the 2 position (C22 α-unit) for Mycobacterium diernhoferi and Mycobacterium sp. strain 3707, a chromogenic rapid grower; (ii) C72 to C78 (av. cn. 75), dienoic with both C22 and C24 α units, containing a small or a large amount of odd-carbon-numbered molecules, for M. vaccae, M. rhodesiae, and M. phlei (chromogenic rapid growers); (iii) C72 to C80 (av. cn. 75 to 77), dienoic with C24 α-unit, containing a moderate or a large amount of odd-carbon-numbered molecules, for M. smegmatis, M. chitae, M. chelonae (M. chelonei), and M. fortuitum (nonchromogenic rapid growers); (iv) C78 to C82 (av. cn. 80), even-carbon-numbered dienoic with C24 α unit for M. agri and M. thermoresistibile (rapid growers); (v) C75 to C81 (av. cn. 77 to 79), odd-carbon-numbered dienoic with C24 α unit for M. nonchromogenicum complex (M. nonchromogenicum, M. terrae, and 'M. novum') (slow growers); (vi) C76 to C84 (av. cn 79 to 81), even-carbon-numbered dienoic with C24 α unit for MAIS ccomplex including M. scrofulaceum, M. avium, and M. intracellulare (slow growers); (vii) C72 to C80 (av. cn. 77 to 79), even-carbon-numbered dienoic with C24 α unit for M. szulgai, M. gordonae, and M. kansasii (chromogenic slow growers); and (viii) C76 to C86 (av. cn. 79 to 81), even-carbon-numbered dienoic with C26 α unit for M. bovis Ravenel and BCG and M. tuberculosis H37Rv. This study demonstrated that gas chromatography-mass spectrometric analysis of the molecular species composition of α-mycolic acid can give rapid, important, and very precise information for the identification of pathogenic and nonpathogenic mycobacterial species.