Hyphenated and comprehensive liquid chromatography × gas chromatography–mass spectrometry for the identification of Mycobacterium tuberculosis

•Comprehensive LC × GC–MS provides more information on the lipid biomarkers present in sputum.•LC × GC–MS results in an enhanced resolution between biomarkers and other lipids in biofluids.•LC coupled to GC–MS provides a better sensitivity for the detection of Mycobacterium tuberculosis.

Tuberculosis is one of the world's most emerging public health problems, particularly in developing countries. Chromatography based methods have been used to tackle this epidemic by focusing on biomarker detection. Unfortunately, interferences from lipids in the sputum matrix, particularly cholesterol, adversely affect the identification and detection of the marker compounds. The present contribution describes the serial combination of normal phase liquid chromatography (NPLC) with thermally assisted hydrolysis and methylation followed by gas chromatography–mass spectrometry (THM–GC–MS) to overcome the difficulties of biomarker evaluation. The in-series combination consists of an LC analysis where fractions are collected and then transferred to the THM–GC–MS system. This was either done with comprehensive coupling, transferring all the fractions, or with hyphenated interfacing, i.e. off-line multi heart-cutting, transferring only selected fractions. Owing to the high sensitivity and selectivity of LC as a sample pre-treatment method, and to the high specificity of the MS as a detector, this analytical approach, NPLC × THM–GC–MS, is extremely sensitive. The results obtained indicate that this analytical set-up is able to detect down to 1 × 103 mycobacteria/mL of Mycobacterium tuberculosis strain 124, spiked in blank sputum samples. It is a powerful analytical tool and also has great potential for full automation. If further studies demonstrate its usefulness when applied blind in real sputum specimens, this technique could compete with the current smear microscopy in the early diagnosis of tuberculosis.