Development and optimization of a gas chromatography/mass spectrometry method for the analysis of thermochemolytic degradation products of phthiocerol dimycocerosate waxes found in Mycobacterium tuberculosis.
Bottom Line: The extraction of PDIMs from culture is simple, and their thermochemolysis is carried out automatically online, thus avoiding the time-consuming derivatization steps of hydrolysis and esterification, usually performed offline.For standard PDIMs in petroleum ether, our optimized method gave an excellent linearity (R(2) = 0.99) at concentrations between 0.172 and 27.5 ng/mL, a good precision (RSD = 11.42%), and a limit of detection (LOD) of 100 pg/mL.For the PDIMs extracted from dilutions of M. tuberculosis culture, the method gave good linearity (R(2) = 0.9685) and an estimated LOD of 400 CFU/mL (CFU = colony forming units) in sterile distilled water.
Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.Show MeSH
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Mentions: The THM-GC/MS(SIM) method was applied to detect the presence of PDIMs extracted from dilutions of M. tuberculosis culture. The SIM chromatograms of the fragment ions, m/z 101 and 88, in Fig. 8 were obtained via thermochemolysis of 50 μL of the petroleum ether extract from 200 μL of a culture dilution in SDW, containing a total of 560 CFU. This is equivalent to 2800 CFU/mL, the lowest concentration of culture analysed in this study. When applied to the petroleum ether extracts from culture dilutions, the method presented good linearity, with correlation coefficients R2 = 0.9685 (C29/C30) and 0.9679 (C32), as illustrated by the results based on duplicate measurements shown in the graphs of Fig. 9. The fact that the calibration graphs for culture do not pass through the origin could be due to the difficulty in preparing homogenous dilutions of M. tuberculosis, because of the clumping nature of the mycobacterial culture. It may also suggest that the petroleum ether extraction of the PDIM waxes from high concentration dilutions is not as efficient as it is for the lower dilutions of culture. The procedural blank, consisting of a petroleum ether extraction applied to 200 μL of SDW, resulted in a clean chromatogram, with no significant areas measured in the retention time window for the target peaks, m/z 101 and 88.
Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.