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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

Extracted ion chromatograms (EICs) obtained following the thermochemolysis of 6.8 ng PDIMs standard for: (a) the fragment ions at m/z 88 and 101, and the molecular ions at m/z 452, 466 and 494; (b) Insert: EICs for the molecular ions at m/z 452 (C29), 466 (C30), and 494 (C32). Note: m/z 452 and 466 refer to both peaks in the overlapping doublets for the C29/C30 methyl mycocerosates.
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fig02: Extracted ion chromatograms (EICs) obtained following the thermochemolysis of 6.8 ng PDIMs standard for: (a) the fragment ions at m/z 88 and 101, and the molecular ions at m/z 452, 466 and 494; (b) Insert: EICs for the molecular ions at m/z 452 (C29), 466 (C30), and 494 (C32). Note: m/z 452 and 466 refer to both peaks in the overlapping doublets for the C29/C30 methyl mycocerosates.

Mentions: The mass spectrum of methyl mycocerosates was generated by analyzing a 50 μL aliquot of a 137.5 ng/mL standard solution of PDIMs in petroleum ether. Hence, 6.8 ng of PDIMs in total were submitted to the thermochemolytic GC/MS method, as described in the Experimental section. Alkaline thermochemolysis causes racemization of the 2-methyl-branched mycocerosates, to give a mix of diastereoisomers due to the presence of the methyl-branched centre at carbon-4. The diastereoisomers from each mycocerosate are partially separable on the GC column, and provide very characteristic doublets,17 as observed in the chromatogram shown in Fig. 2, generated by extracting the fragment ions of m/z 88 and 101 from the full scan mass spectra. The earlier eluting compounds are the trimethylhexacosanoic acid and the tetramethyl hexacosanoic acid methyl esters, whose individual doublets overlap to generate the typical composite C29/C30 doublet peak. The tetramethyloctacosanoic acid methyl ester (C32), whose diastereoisomers produce the second doublet peak, follows 30 s later. A much less pronounced doublet is observed for each of the molecular ions monitored at m/z 452 (C29), m/z 466 (C30), and m/z 494 (C32). The chromatographic profile for the molecular ions is amplified in Fig. 2(b), and Fig. 3 presents the corresponding fragmentation patterns obtained for the mycocerosic acid methyl esters released from 6.8 ng total PDIMs.


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)

Extracted ion chromatograms (EICs) obtained following the thermochemolysis of 6.8 ng PDIMs standard for: (a) the fragment ions at m/z 88 and 101, and the molecular ions at m/z 452, 466 and 494; (b) Insert: EICs for the molecular ions at m/z 452 (C29), 466 (C30), and 494 (C32). Note: m/z 452 and 466 refer to both peaks in the overlapping doublets for the C29/C30 methyl mycocerosates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: Extracted ion chromatograms (EICs) obtained following the thermochemolysis of 6.8 ng PDIMs standard for: (a) the fragment ions at m/z 88 and 101, and the molecular ions at m/z 452, 466 and 494; (b) Insert: EICs for the molecular ions at m/z 452 (C29), 466 (C30), and 494 (C32). Note: m/z 452 and 466 refer to both peaks in the overlapping doublets for the C29/C30 methyl mycocerosates.
Mentions: The mass spectrum of methyl mycocerosates was generated by analyzing a 50 μL aliquot of a 137.5 ng/mL standard solution of PDIMs in petroleum ether. Hence, 6.8 ng of PDIMs in total were submitted to the thermochemolytic GC/MS method, as described in the Experimental section. Alkaline thermochemolysis causes racemization of the 2-methyl-branched mycocerosates, to give a mix of diastereoisomers due to the presence of the methyl-branched centre at carbon-4. The diastereoisomers from each mycocerosate are partially separable on the GC column, and provide very characteristic doublets,17 as observed in the chromatogram shown in Fig. 2, generated by extracting the fragment ions of m/z 88 and 101 from the full scan mass spectra. The earlier eluting compounds are the trimethylhexacosanoic acid and the tetramethyl hexacosanoic acid methyl esters, whose individual doublets overlap to generate the typical composite C29/C30 doublet peak. The tetramethyloctacosanoic acid methyl ester (C32), whose diastereoisomers produce the second doublet peak, follows 30 s later. A much less pronounced doublet is observed for each of the molecular ions monitored at m/z 452 (C29), m/z 466 (C30), and m/z 494 (C32). The chromatographic profile for the molecular ions is amplified in Fig. 2(b), and Fig. 3 presents the corresponding fragmentation patterns obtained for the mycocerosic acid methyl esters released from 6.8 ng total PDIMs.

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