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Determination of Porothramycin by Ultra-High-Performance Liquid Chromatography
Kettnerová, Eliška ; Nesměrák, Karel (advisor) ; Bosáková, Zuzana (referee)
Porothramycin is the antibiotic of anthramycin group and is produced by Streptomyces albus. Porothramycin is characterized by antibacterial and especially antitumor activity. But it has also been proved to be cardiotoxic; and therefore, it has not been applied in clinical practice yet. However, some genes involved in the synthesis of porothramycin can be used for the preparation of new nontoxic derivatives of antibiotics by means of combinatorial biosynthesis. For this purpose, it was necessary to develop a new routine chromatographic UHPLC-UV- MS method for analysis of porothramycin. A suitable cultivation broth for production of porothramycin was found using disk diffusion test with the sensitive strain Kocuria rhizophila. Then, 1.7 mg of porothramycin standard, which was not available on the market, was isolated from the cultivation broth and used for the development and validation of the method. Also, it was found out that apart from porothramycin, two porothramycin-related compounds were produced into the cultivation broth. In the final method for determination of porothramycin, the solid phase extraction using styren-divinylbenzene sorbent with vinylpyrrolidone functionalities was applied. The extracts were then loaded onto the UHPLC Acquity BEH C18 column and analyzed under gradient mode...
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Isolation and characterisation of biologically active substances
Kettnerová, Eliška ; Nesměrák, Karel (advisor) ; Musilová, Adéla (referee)
This Bachelor thesis aims at isolation and partial identification of biologically active substances which are produced by actinomycetes and can be potentially applied in medicine. Cultivation broths of actinomycetes containing their metabolites were purified and pre-concentrated by solid phase extraction. Then, the bioassay of the extracts by Kirby-Bauer test using the sensitive strain Kocuria rhizophila was performed. Biologically active metabolites were analyzed and isolated by ultra- performance liquid chromatography with photo diode array detector. Isolated substances were assayed by mass spectrometry, which yielded relative molecular mass values of the unknown compounds. The values were compared with relative molecular masses of compounds listed in a chemical database, which involves natural products including antibiotics. We revealed that the unknown biologically active substances do not refer to any already discovered compound present in the database suggesting that the unknown compounds may be novel. More mass spectrometry and nuclear resonance experiments have to be carried out in order to elucidate their structure. Key words: actinomycetes, antibiotics, SPE, UPLC, HPLC Subject heading: analysis of secondary metabolites, bioassay test, isolation of biologically active compounds,...
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