A study of online and offline derivatization methods for tuberculostearic acid - a biomarker for M. Tuberculosis

Mycobacteria such as M. tuberculosis have the ability of producing lipid components specific to their cell wall. The detection of these lipids can serve to diagnose tuberculosis, a contagious disease that still affects millions of people worldwide. Microscopic examination of sputum can give a quick diagnosis of pulmonary M. tuberculosis but it lacks sensitivity, while culturing may need up to 8 weeks for a definitive result. 10-R-Methyloctadecanoic acid or TBSA, a saturated methyl branched fatty acid, is one of the lipids found in the cell wall of mycobacteria and of certain related bacterial species in the order of Actinomycetales. In M. tuberculosis, TBSA may represent up to 25% of the total fatty acid content and has been identified in the sputum of TB infected patients, by using gas chromatography-mass spectrometry GC-MS. Complex sample extraction and preparation procedures are usually necessary prior to separating the derivatized analytes on the GC column. In the present work, aliquots of pooled positive and pooled negative sputum samples were analyzed by: (i) direct thermochemolysis of sputum deposit aliquots; (ii) thermochemolysis of TBSA extracted from sputum deposits, and (iii) the injection in the GC inlet of the boron trifluoride alkylated derivatives obtained from the sputum extracts. The derivatized compounds were separated on a DB-5MS capillary column and detected by a quadrupole mass spectrometer with electron impact ionisation source. The study discusses the implications of the various derivatization methods on the GC-MS response in selected ion monitoring, SIM mode, for the molecular ion of the methylated TBSA. The yield of TBSA extracted from sputum and submitted to TMAH derivatisation is only a fraction of that obtained from the direct thermochemolytic analysis of the vortexed deposit, but the extraction of TBSA also reduces the risk of overloading the GC capillary column, drops the baseline and improves the peaks shape.