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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.

Nicoara SC, Minnikin DE, Lee OC, O'Sullivan DM, McNerney R, Pillinger CT, Wright IP, Morgan GH - Rapid Commun. Mass Spectrom. (2013)

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.

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Related in: MedlinePlus

SIM chromatograms showing the target fragment ions m/z 101 (top trace) and 88 (bottom trace) resulting from the cleavage of the major trimethyl- (C29) and tetramethyl- (C30 and C32) methyl mycocerosates obtained by the thermochemolysis of 137.5 pg total PDIMs standard, from an aliquot of 50 μL of a 2.75 ng/mL solution.
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fig05: SIM chromatograms showing the target fragment ions m/z 101 (top trace) and 88 (bottom trace) resulting from the cleavage of the major trimethyl- (C29) and tetramethyl- (C30 and C32) methyl mycocerosates obtained by the thermochemolysis of 137.5 pg total PDIMs standard, from an aliquot of 50 μL of a 2.75 ng/mL solution.

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.


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.

Nicoara SC, Minnikin DE, Lee OC, O'Sullivan DM, McNerney R, Pillinger CT, Wright IP, Morgan GH - Rapid Commun. Mass Spectrom. (2013)

SIM chromatograms showing the target fragment ions m/z 101 (top trace) and 88 (bottom trace) resulting from the cleavage of the major trimethyl- (C29) and tetramethyl- (C30 and C32) methyl mycocerosates obtained by the thermochemolysis of 137.5 pg total PDIMs standard, from an aliquot of 50 μL of a 2.75 ng/mL solution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3824232&req=5

fig05: SIM chromatograms showing the target fragment ions m/z 101 (top trace) and 88 (bottom trace) resulting from the cleavage of the major trimethyl- (C29) and tetramethyl- (C30 and C32) methyl mycocerosates obtained by the thermochemolysis of 137.5 pg total PDIMs standard, from an aliquot of 50 μL of a 2.75 ng/mL solution.
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.

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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physical Sciences, Faculty of Science, The Open University, Walton Hall, Milton Keynes, MK7 6AA, UK.

Show MeSH
Related in: MedlinePlus