Title: The development, verification and objectives of a mycocerosic acid based HPLC-HRMS method for TB diagnostics in paleopathology

Authors: Orsolya Anna Váradi^1,2, Olga Spekker¹, Heidi Y. Jäger³, Frank Maixner³, Albert Zink³, Dávid Rakk², Csaba Vágvölgyi², Gabriella Terhes⁴, Enikő Szvák⁵, Ildikó Pap^1,5, Ildikó Szikossy⁵, György Pálfi¹, András Szekeres², David E. Minnikin⁶

Affiliations: ¹Department of Biological Anthropology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; ²Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; ³Institute for Mummy Studies, Eurac Research, Bolzano, Italy; ⁴Institute of Clinical Microbiology, Faculty of Medicine, University of Szeged, Szeged, Hungary; ⁵Department of Anthropology, Hungarian Natural History Museum, Budapest, Hungary; ⁶Institute of Microbiology and Infection, School of Biosciences, University of Birmingham, Birmingham, UK

Abstract: The diagnosis of ancient mycobacterial disease in archaeological material requires a multidisciplinary approach. Beside aDNA analyses, a variety of analytical methods have been employed for mycobacterial lipid biomarker profiling. Most commonly mycolic acids (MAs), mycocerosic acids (MCs), and phthiocerols are monitored to detect ancient tuberculosis (TB). For MA and phthiocerol analysis, a well-developed, long-existing method is used in paleopathological investigations, with the application of HPLC coupled with fluorescence detector. For the analysis of MCs, mass spectrometry has been traditionally applied, using selected ion monitoring, negative ion-chemical ionisation GC-MS for the detection of pentafluorobenzyl ester derivatives.

Our aim was to develop and optimise a HPLC-MS method for the detection of MAs and MCs. In contrast with aDNA, obviously there is no option for amplifying the extracted lipid biomarkers; thus, the optimisation of the extraction was surpassingly important. To have a reference for later analysis of archaeological samples, a lipid profile library, regarding both MAs and MCs, had to be built as the next step. In the development of the library, a variety of tuberculous and non-tuberculous strains were included. When the optimised detection method and the lipid profile library seemed sufficient to confirm TB-infected cases among archaeological human remains based on MC analysis, it was successfully tested on samples taken from Vác mummies.

In the first part of our presentation, we would like to briefly introduce the above-mentioned procedure and its main results. In the second part, we would like to start a conversation about the objectives and possibilities, which could lead to the further development of the method, and hopefully, to the expansion of the application.