Development of sample clean up methods for the analysis of Mycobacterium tuberculosis methyl mycocerosate biomarkers in sputum extracts by gas chromatography-mass spectrometry.
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.
Centre for Earth, Planetary, Space and Astronomical Research CEPSAR, The Open University, Milton Keynes, UK. Electronic address: simona.nicoara@open.ac.uk.
Simona C
SC
Nicoara
Life, Health and Chemical Sciences, The Open University, Milton Keynes, UK.
Nicholas W
NW
Turner
Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, UK.
David E
DE
Minnikin
Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham, UK.
Oona Y-C
OY
Lee
London School of Hygiene & Tropical Medicine, London, UK.
Denise M
DM
O'Sullivan
London School of Hygiene & Tropical Medicine, London, UK.
Ruth
R
McNerney
Biomedical Research and Training Institute, Harare, Zimbabwe.
Reggie
R
Mutetwa
Biomedical Research and Training Institute, Harare, Zimbabwe.
Liz E
LE
Corbett
Centre for Earth, Planetary, Space and Astronomical Research CEPSAR, The Open University, Milton Keynes, UK.
Geraint H
GH
Morgan
D000942:Antigens, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D015415:Biomarkers / Q000032:analysis* / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D008055:Lipids / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D054802:Molecular Imprinting; D009169:Mycobacterium tuberculosis / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000379:methods*; D013183:Sputum / Q000382:microbiology*
D000942:Antigens, Bacterial; D015415:Biomarkers; D008055:Lipids; C008901:phthiocerol dimycocerosate
Cholesterol; GC–MS; Molecularly imprinted polymers; Mycobacterium tuberculosis; Solid phase extraction; Thermochemolysis