Compounds library verification using high-pressure liquid chromatography coupled with mass spectometry

Skozi razvoj in potrebo po čim krajšem in učinkovitem iskanju novih zdravilnih učinkovin je v ospredje stopila zmožnost sinteze knjižnic strukturno različnih spojin. Za odkrivanje novih zdravilnih učinkovin se trenutno najpogosteje uporablja metodo visoke zmogljivosti rešetanja (High Throughput Screening – HTS), iz katere so se na osnovi načrtovanja strukture in lastnosti molekul razvili še drugi sorodni koncepti sintezne kemije. V zadnjem desetletju se je tako znatno povečalo število novih odkritih spojin, vendar se kljub temu še vedno poslužujemo klasičnega pristopa rešetanja, zato poleg HTS knjižnic uporabljamo tako imenovane hišne (in-house) knjižnice. Namen magistrske naloge je bil razviti in optimizirati metodo tekočinske kromatografije visoke ločljivosti (HPLC) sklopljene z masnim detektorjem za identifikacijo ter kvantitativno določanje spojin knjižnice Fakultete za farmacijo. Glavni poudarek je bil na razvoju standardne HPLC metode, ter na velikem številu pripravljenih spojin, na katerih smo preverili učinkovitost naše metode. Predvsem smo raziskali vpliv različnih kromatografskih pogojev, kot je sestava mobilne faze, izbira primerne kromatografske kolone ter izbira ustreznega gradienta, kar bi omogočalo univerzalno uporabo. Z uporabo HPLC/MS metode smo bili sposobni identificirati 80 % preiskovanih spojin. Metoda je tako primerna za kvalitativno in kvantitativno vrednotenje. Ključni del raziskovalnega dela je bilo ročno preverjanje pridobljenih rezultatov, saj sodobno metode še ne omogočajo avtomatiziranega vrednotenja tako pridobljenih podatkov. Podatke smo ovrednotili in identificirali za osnovno spojino tudi prisotne nečistote. Z uporabo masne spektrometrije smo lahko tudi predvideli, kaj se je s preiskovano spojino zgodilo ter katere kemijske reakcijo so potekle pred ali po analiziranju spojin. Ostalim 20 % preiskovanih spojin niso ustrezali kromatografski pogoji, zato bi jih bilo potrebno obravnavati individualno.Focus on the ability of synthesize libraries of structurally different compounds was given through discovery and need for quick and effective search of new active substances. For the discovery of new active substances, is most commonly used the High Throughput Screening (HTS) method, from which other related concepts of synthetic chemistry have been developed based on the design of the structure and properties of molecules. In the last decade, the number of new detected compounds has increased significantly, but nevertheless, we continue to use the classic approaches. In addition to HTS libraries we use the so-called in-house libraries. The aim of this thesis was to develop and optimize HPLC method coupled with a mass detector to identify and quantify the compounds from the library of Faculty of pharmacy. The main focus was on the development of the standard HPLC method, and on the large number of prepared compounds, for which we checked the effectiveness of our method. In particular, we investigated the impact of various chromatographic conditions, such as the composition of the mobile phase, the choice of an appropriate chromatographic column, and the choice of the appropriate gradient, which would allow the universal use. Using the HPLC / MS method, we were able to identify 80% of the investigated compounds. Method is therefore suitable for qualitative and quantitative evaluation. A key part of the research work was to manually verify the results obtained, since modern methods do not allow automated evaluation of data. The data were statistically evaluated and identified for the main compound and also its disintegrating products. By using the mass spectrometry, we could also predict what happened to the investigated compound and which chemical reaction has occurred before or after analyzing the compounds. The chromatographic conditions were not suitable for the remaining 20% of the investigated compounds, thus should be considered individually