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

Simona C. Nicoara, Nicholas W. Turner, David E. Minnikin, Oona Y.-c. Lee, Denise M. O'sullivan, Ruth Mcnerney, Reggie Mutetwa, Liz E. Corbett, Geraint H. Morgan

Research output: Contribution to journalArticlepeer-review

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Abstract

A proof of principle gas chromatography–mass spectrometry method is presented, in combination with clean up assays, aiming to improve the analysis of methyl mycocerosate tuberculosis biomarkers from sputum. Methyl mycocerosates are generated from the transesterification of phthiocerol dimycocerosates (PDIMs), extracted in petroleum ether from sputum of tuberculosis suspect patients. When a high matrix background is present in the sputum extracts, the identification of the chromatographic peaks corresponding to the methyl derivatives of PDIMs analytes may be hindered by the closely eluting methyl ether of cholesterol, usually an abundant matrix constituent frequently present in sputum samples. The purification procedures involving solid phase extraction (SPE) based methods with both commercial Isolute-Florisil cartridges, and purpose designed molecularly imprinted polymeric materials (MIPs), resulted in cleaner chromatograms, while the mycocerosates are still present. The clean-up performed on solutions of PDIMs and cholesterol standards in petroleum ether show that, depending on the solvent mix and on the type of SPE used, the recovery of PDIMs is between 64 and 70%, whilst most of the cholesterol is removed from the system. When applied to petroleum ether extracts from representative sputum samples, the clean-up procedures resulted in recoveries of 36–68% for PDIMs, allowing some superior detection of the target analytes.
Original languageEnglish
Pages (from-to)135-142
JournalJournal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Volume986-987
Early online date16 Jan 2015
DOIs
Publication statusPublished - 1 Apr 2015

Keywords

  • Cholesterol
  • Mycobacterium tuberculosis
  • Solid phase extraction
  • Molecularly imprinted polymers
  • Thermochemolysis
  • GC–MS

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