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A derivatization protocol for mycolic acids detection using liquid chromatography/mass spectrometry




Zurita Urrea, Paulina, author
Belisle, John T., advisor
Jones, Robert, committee member
Salman, Mo, committee member

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New tools for the diagnosis and control of Tuberculosis are major challenges. In this context the use of biomarkers can be applied for detecting characteristic signatures from the tuberculosis-infected host and the pathogen. Mycolic acids are considered as a hallmark of the Mycobacterium genus being abundant in the mycobacterial cell wall. In this study a derivatization protocol was tested to enhance the detection of mycolic acid after the attachment of a quaternary amine and analysis of the derivatized products in the positive ionization mode with liquid chromatography/mass spectrometry. Three groups were considered i) mycolic acid standard ii) human urine spiked with mycolic acid standard, and iii) human serum spiked with mycolic acid standard. Each group included the analysis of a set of non-derivatized mycolic acids in positive and negative ionization mode, and derivatized mycolic acids in positive mode. The derivatization process applied to the mycolic acid standard and to the urine samples spiked with mycolic did not improve the ion volume value compared to the respective non-derivatized samples. Serum samples, however, showed a significant enhancement in the ion volume of the different mycolic acids analyzed compared to the non-derivatized serum samples (α=0.05). The method detection limit for the three groups was also achieved. Urine and serum samples spiked with mycolic acids showed higher detection limits compared to the mycolic acid standard; this was expected due the lipid extraction protocol and the complex nature of these fluids. The derivatization protocol did not improve the method detection limit compared to the non-derivatized samples. The overall results make the derivatization protocol questionable to be applied routinely in biological samples. However, the results obtained after the derivatization of serum samples could point to the advantages of using a derivatization protocol to study possible interactions between mycolic acids and other molecules present in serum that could be impeding their detection.


2012 Fall.
Includes bibliographical references.

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mass spectrometry
mycolic acids


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