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 extracted ion chromatograms generated for the methylated phthiocerol A components from 6.8 ng of PDIMs are illustrated in Fig. 4(a). These di-methylated derivatives of the main phthiocerol A components C34 and C36, elute at high oven temperatures of 348 °C and 350 °C, respectively, which increased the thermal stress for the DB-5MS column used, and also extended the analysis time to 21 min. The chromatographic profiles are shown for the fragment ions at m/z 201 (grey trace) and the higher mass fragment ions of m/z 339 and 367 (black traces). Their 70 eV EI molecular ions, expected at m/z 554 (methylated C34) and 582 (methylated C36), were not observed in the mass spectra.
Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.