HPLC (High Performance Liquid Chromatography)HPLC is a powerful chromatography technique capable of separating similar substances from a mixture in a short time. The method requires a mobile phase (liquid) and a stationary phase (liquid/solid). The stationary phase is usually indicated by the column used, while the eluent is the mobile phase. The two phases are selected to best suit the sample and the purpose of the separation. The resolution and order of elution depends on the stationary and mobile phase selected. Substances that have a greater affinity for the mobile phase will emerge first, while others with an affinity for the stationary phase will remain in the column longer (Meyer, 2010). In order to force the mobile phase into the column filled with very small particles, a constant high pressure is required to be applied. The basic principle is that under the same conditions, the time between the injection of the component and its elution remains constant. The result is a graph showing changes over time in signal intensities resulting from the separation of substances. The technique can be used in both qualitative and quantitative approaches, as the height and area of ​​each peak are proportional to the concentration of the corresponding substance (Meyer, 2010). Mycolic acids can be recognized based on their chain length (C70 - C90). , the presence of double bonds, their long side chain, their additional oxygen or methyl functions. The three main mycobacterial mycolate classes can be separated by “normal phase” HPLC, but the characteristic peaks of MTBC cannot be identified. However, “reserve phase” HPLC (rpHPLC), can separate mycolic acids based on their chain length and paper length......, D. (1999) Genotypic analysis of Mycobacterium tuberculosis from medieval human remains, Microbiology 145: 899-904;22. Scorpio, A., Zhang, Y. (1996) Mutations in pncA, a gene encoding pyrazinamidase/nicotinamidase, cause resistance to the antituberculosis drug pyrazinamide in tubercle bacilli, Nat Med 2(6): 662-667;23 . Zimhony, O., Cox, JS, Welch, JT, Vilcheze, C. and Jacobs, WR, Jr. (2000) Pyrazinamide inhibits eukaryotic fatty acid synthetase I (FASI) of Mycobacterium tuberculosis, Nat Med 6: 1043 –1047;24. Hofreiter, M., Serre, D., Poinar, H.N., Kuch, M., Pääbo, S. (2001) Ancient DNA, Nat Rev 2(353): 353-359;25. Shi, W., Zhang, X., Jiang, X., Ruan, H. et al. (2011) Pyrazinamide inhibits trans-translation in Mycobacterium tuberculosis: a potential mechanism to shorten the duration of anti-tuberculosis chemotherapy, Science 333(6049): 1630–1632.