Rapid determination of growth inhibition of Mycobacterium tuberculosis by GC-MS/MS quantitation of tuberculostearic acid

Classical determination of growth inhibition of Mycobacterium tuberculosis in macrophages and mice by new candidate anti-TB drugs utilizes the determination of colony forming units (CFUs) from lung homogenates, a labor-intensive process requiring 2-3 weeks incubation. Qualitative analysis of tuberculostearic acid (TBSA), a cell wall associated biomarker found in M. tuberculosis, has been investigated for clinical diagnosis of tuberculosis (TB) but few reports exist of attempts to quantitate TBSA. Gas chromatography-mass spectroscopy (GC-MS/MS) was used in quantitating the derivatized methyl ester of TBSA during growth of M. tuberculosis in axenic medium, macrophage cultures and in the lungs of gamma interferon knockout (GKO) mice with and without exposure to anti-TB agents. The quantity of TBSA methyl ester (TBSAME) in the absence of and following exposure to anti-TB drugs was positively correlated with CFU in all three models. The stability of TBSA precludes its use as a surrogate for bactericidal activity but its exceptional thermal stability enables lung homogenates to be autoclaved prior to analysis. GC-MS/MS determination of TBSA is a rapid, sensitive and accurate means of detecting growth inhibition of any strain of M. tuberculosis in cell culture and in vivo.