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: For the development and optimization of the analytical method, we focused on the GC/MS(SIM) response obtained for the fragment ions m/z 101 and 88, each giving the two characteristic doublet peaks in C29/C30 and C32, in the retention time window between 16.4 and 17.3 min. A typical extracted ion chromatogram for these two fragment ions, for a solution of PDIMs of concentration 2.75 ng/mL of standard in petroleum ether, is shown in Fig. 5. This concentration is similar to that obtained for the PDIMs released in most of the sputum extracts in 1 mL of petroleum ether. For quantitative analysis, the m/z 101 doublet peak area was measured at both retention times, using the manual integration facility in the Agilent ChemStation software. Daily runs of the standard of PDIMs allowed for retention time alignment and correction, whenever chromatographic drift occurred. Each programmable parameter of the PTV inlet was studied to optimize the GC/MS(SIM) response for a solution of PDIMs of 2.75 ng/mL concentration in petroleum ether, hence for 137.5 pg of standard per liner.
Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.