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

Tuberculosis is one of the world's most emerging public health problems, particularly in developing countries. Chromatog. 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 compds. The present contribution describes the serial combination of normal phase liq. chromatog. (NPLC) with thermally assisted hydrolysis and methylation followed by gas chromatog.-​mass spectrometry (THM-​GC-​MS) to overcome the difficulties of biomarker evaluation. The in-​series combination consists of an LC anal. 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 anal. approach, NPLC × THM-​GC-​MS, is extremely sensitive. The results obtained indicate that this anal. 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 anal. 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.