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

Abstract

RATIONALE: The phthiocerol dimycocerosates (PDIMs) are certain stable and hydrophobic waxes found in the cell membrane of Mycobacterium tuberculosis, bacteria that cause an infectious disease of growing concern worldwide. Previous studies report the analysis of derivatives of the hydrolysed PDIMs from biological samples, following complex extraction and offline derivatization of PDIMs biomarkers, prior to their analysis by gas chromatography/mass spectrometry (GC/MS).METHODS: We developed and optimized a GC/MS method based on selected ion monitoring (SIM) to detect the derivatives produced via the thermally assisted hydrolysis and methylation (THM) of the PDIMs from the cell membrane of M. tuberculosis. 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.RESULTS: For standard PDIMs in petroleum ether, our optimized method gave an excellent linearity (R2 = 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 (R2 = 0.9685) and an estimated LOD of 400 CFU/mL (CFU = colony forming units) in sterile distilled water.CONCLUSIONS: A GC/MS(SIM) method is presented for the rapid and quantitative detection of M. tuberculosis, based on the online thermochemolysis of lipidic biomarkers extracted from the bacterial culture. The method has the potential to be applied in human and veterinary clinical laboratories for the rapid diagnosis of tuberculosis in infected biological samples. © 2013 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons, Ltd.

Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography–mass spectrometry

“…4 a. The doublet peaks can be observed, which are characteristic to the coeluting diastereoisomers of methyl mycocerosates C 29 and C 30 , and to the diastereoisomers of C 32 , respectively, as discussed in our earlier publications [14,15] . The mass spectra of C 29 , C 30 , and C 32 are shown in Fig.…”

“…The top line of Fig. 3 shows the current methodologies as described in our prior publications [14,15] . The ion chromatograms for m / z 101 and m / z 88 obtained for the methyl derivatives of 140 pg of PDIMs standard are shown in Fig.…”

“…2 , were then analyzed by GC–MS. The initial THM-GC–MS method development [14] and its application to a batch of positive and negative real sputum samples [15] were presented in our earlier publications, which focused on the overall method performance without reporting and discussing the particular results obtained in certain sputum sample extracts with a high level of matrix compounds. Blind analysis of the PE extracts of 400+ sputum specimens [15] , using our THM-GC–MS method gave 64.9% sensitivity and 76.2% specificity, and it was noted that other components of sputum, such as cholesterol, may hinder the analysis.…”

GCMS Based Detection of Lipid Biomarkers of Mycobacterium tuberculosis in the Serum Specimen

“…The presence of these molecules could be attributed to presence of PDIMs. Gas chromatographic-mass spectrometric (GC-MS) analysis has proven to be successful for the TB diagnosis by detecting M. tuberculosis PDIMs biomarkers in sputum [9,21,22] . This PDIM has an exceptionally low polarity hence it is an advantage to separate these molecules from other host specific lipids.…”

Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS

“…For example, Minnikin et al ( 10,(24)(25)(26) and Daffe et al ( 9,(27)(28)(29) used GC/MS analysis together with NMR spectroscopy for complete characterization of DIMs, following extraction steps, chromato graphic separations, and chemical reactions. Recently, MALDI-TOF ( 16,29,30 ) and ESI-Fourier transform ion cyclotron resonance ( 31 ) MS have also been applied for profi ling PDIM and PGLs.…”