A comparison of four sputum pre-extraction preparation methods for identifying and characterising Mycobacterium tuberculosis using GCxGC-TOFMS metabolomics

Abstract

In many pulmonary diseases, sputum is a valuable sample material for use in disease characterisation and diagnostics. However, due to its high viscosity and uneven consistency (lumpiness), it is difficult to obtain reproducible/repeatable results during compound extraction and analysis. We subsequently investigated and compared four sputum pre-extraction preparation methods using: 1) Sputolysin; 2) a combination of N-acetyl-l-cysteine and sodium hydroxide (NALC-NaOH); 3) NaOH alone, and 4) a simple ethanol homogenisation method, prior to sputum extraction and metabolomics analyses. The simple ethanol homogenisation approach proved to be the comparatively superior sputum pre-extraction preparation method, considering its repeatability, the number of characteristic compounds extracted, its ability to extract those compounds best differentiating the sample groups (Mycobacterium tuberculosis-spiked and clinically confirmed TB-positive patient samples from each of the controls respectively), and its detection limit. This developed methodology subsequently allows for accurate GC based analyses of sputum, and hence, could contribute significantly to the better characterisation or diagnostics of not only tuberculosis, but also potentially other pulmonary diseases, including, interstitial lung disease, cystic fibrosis, lung cancer, pneumonia and any other bacterial induced pulmonary diseases producing sputum.