Mycolic acids (MAs), which play a crucial role in the architecture of mycobacterial cell walls, were analyzed
using electrospray ionization tandem mass spectrometry. A targeted analysis based on the 10 most abundant
and characteristic multiple reaction monitoring pairs was used to profile the crude fatty acid mixtures from
Mtb and several nontuberculous mycobacterial strains. Comparative analysis yielded unique profiles for MAs,
enabling the reliable identification of mycobacterial species. In a case-control study of tuberculosis (TB) and
non-TB Polish patients, we demonstrated the potential diagnostic utility of our approach for the rapid
diagnosis of active TB with sensitivity and specificity surpassing those of existing methods. This robust
method allows the identification of TB-positive patients after 2 h of sample preparation in the case of direct
sputum analysis or 10 days of culturing, both of which are followed by 1 min of liquid chromatography–
tandem mass spectrometry analysis
