Figure 1.
Structures of the M. tuberculosis C29, C30 and C32 mycocerosic acid methyl esters.
The ions at m/z 452, 466 and 494 correspond to the molecular weights of these esters. Ions at m/z 88 and 101 are characteristic for 2-methyl branched fatty acid methyl esters, the former resulting from a well-known “McLafferty” rearrangement with transfer of one hydrogen atom.
Figure 2.
Thermochemolytic-gas chromatography-electron impact/mass spectrometry (THM-GC-EI/MS) of purified phthiocerol dimycocerosate (PDIM) waxes.
The selected ion monitored EI/MS chromatogram shows the target fragment ions m/z 101 and m/z 88, resulting from the major trimethyl C29 and tetramethyl C30, and C32 mycocerosates released by thermochemolysis of the PDIMs. A minor proportion of a tetramethyl C31 mycocerosate is also indicated. The characteristic doublet peaks are due to racemisation during the alkaline hydrolysis. The method relied on recognizing comparable proportions of the combined C29/C30 peak and the C32 peak, as was the case in all the positive specimens. Some minor variation in the ratios of the individual peaks was observed, but this did not affect the diagnostic ratios.
Figure 3.
THM-GC-EI/MS of sputum spiked with PDIMs.
The graphs show the peak areas obtained for C29/C30 and C32 mycocerosates generated from spiking 0.2 mL of pooled TB negative sputum samples with PDIM standard, (A) in the range 0.14–2.75 ng, and (B) expanding the range between 0.14–27.5 ng.
Figure 4.
THM-GC-EI/MS of sputum spiked with M. tuberculosis bacilli.
The graph shows the peak areas obtained for C29/C30 and C32 mycocerosates generated from spiking 1 mL of pooled TB negative sputum sample with M. tuberculosis culture (140–5,600 CFU/mL).
Figure 5.
THM-GC-EI/MS traces of TB negative sputum spiked with M. tuberculosis bacilli.
The chromatograms show the fragment ions m/z 101 and m/z 88 for the extract from 1 ml sample of TB negative sputum spiked with 140 CFU of M. tuberculosis culture.
Figure 6.
Examples of THM-GC-EI/MS chromatograms from sputum specimens from suspected TB cases.
Chromatograms show the profile of the fragment ion m/z 101 in (A) negative, and positive sputum samples with (B) high, and (C) low amounts of PDIMs present. Note the characteristic doublets for the mycocerosic acid methyl esters C29/C30 and C32 in b–c.
Table 1.
Performance of THM-GC-EI/MS compared to smear microscopy and culture on Lowenstein-Jensen for detection M. tuberculosis in 395 sputum samples.
Table 2.
Secondary analysis of the performance of THM-GC-EI/MS compared to smear microscopy and culture on Lowenstein Jensen.