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Tekočinska kromatografija visoke ločljivosti sklopljena z masno spektrometrijo (HPLC-MS) je tehnika, ki postaja nepogrešljiva na področju farmacije. Združuje dve analitski tehniki, in sicer tekočinsko kromatografijo visoke ločljivosti in zelo občutljivo ter specifično masno spektrometrijo. Tekočinska kromatografija loči komponente zmesi glede na njihovo afiniteto do stacionarne in mobilne faze. Te komponente lahko zazna UV svetloba. Osnova pri masni spektrometriji je ločevanje ioniziranih komponent v vakuumu glede na njihovo razmerje med maso in nabojem. Pri razvoju zdravilnih učinkovin se srečujemo s knjižnicami spojin. Testiramo jih na bioloških tarčah s pomočjo rešetanja visokih zmogljivosti. Biti morajo urejene in dobro oblikovane, da lahko v čim krajšem času testiramo čim večje število spojin. Pomembno je, da so spojine, ko jih shranimo v knjižnico ustrezne kakovosti in v nadaljnje tudi dobro shranjene. Namen magistrske naloge je bil vrednotiti knjižnico spojin, sintetiziranih na Fakulteti za farmacijo s HPLC-UV-MS tehniko. Število vzorcev v knjižnici spojin je bilo 1145. Za separacijo smo uporabili reverzno-fazno kromatografijo. Na masnem spektrometru smo uporabili elektrorazprševalno ionizacijo. Pri vrednotenju rezultatov smo imeli nekaj težav z analizo kromatogramov, na kromatogramih so se pojavili popačeni vrhovi in vrhovi slabe intenzitete. Prav tako je prišlo do napak pri pripravi vzorcev, dekarboksilacije spojin, hidrolize spojin, fragmentacije spojin in nestabilnosti raztopin. Vendar je bila instrumentalna metoda, kljub napakam primerna za večino spojin (90,1 %). Knjižnico smo uredili in v Excel tabeli zbrali vse podatke, ki smo jih pridobili pri interpretiranju rezultatov. Spojinam smo določili retencijski čas, ocenili čistost in odzive pri UV spektroskopiji. Z masno spektrometrijo smo potrdili strukturo spojin, pridobili MS kromatogram in masni spekter spojin. Določili smo nastanek aduktov. S tem smo kvantitativno in kvalitativno ovrednotili knjižnico spojin ter tudi na reprezentativnem vzoru pridobili podatke o obnašanju spojin vodnic v HPLC-UV-MS kromatografskem sistemu. High-pressure liquid chromatography coupled with mass spectrometry is a technique which is becoming indispensable in the pharmaceutical field. It combines two analytical techniques: high-performance liquid chromatography and mass spectrometry. Liquid chromatography separates the components of the compound according to their affinity with the stationary and mobile phase. These components can be detected by UV light. The basis of mass spectrometry is separating ionized components in a vacuum, according to their mass-to-charge ratio. In the development of active substances, compound libraries are one of the key compnents of research. They are tested on various biological targets, using high-throughput screening. They need to be organised and well-formed, to test the largest number of compounds in the shortest possible time. It is of key importance, that the compounds, that are being kept in the library, are of adequate quality and well preserved for future use. The aim of the master's thesis was to assess the library of compounds, which were synthesised at the Faculty of Pharmacy with the HPLC-UV-MS technique. The number of samples in the compound library was 1145. Reverse-phase chromatography was used for the separation and electrospray ionization was used for ionization on the mass spectrometer. Many difficulties were also encountered in the evaluation of results, namely poor mass spectra, decarboxylation of compounds, mistakes in preparation of samples, hydrolysis of compounds, fragmentation of compounds, and instability of solutions. However, despite the errors, the instrumental method was suitable for most of the compounds (90,1%). We organised the library and collected all data, that were gathered when interpreting results, in an Excel table. We determined the retention time of the compounds, assessed the purity and reactions in UV spectroscopy. We confirmed the structure of the compounds with mass spectrometry, attained the MS chromatogram and mass spectra of the compounds. We determined the formation of adducts. This allowed us to quantitatively and qualitatively evaluate the compound library, and also to obtain data on the behaviour of the lead compounds in an HPLC-UV-MS chromatographic system on a representative sample.

Problem lažno pozitivnih zadetkov in zadetkov z neugodnimi lastnostmi za razvoj do spojin vodnic, ki jih dobimo pri rešetanju visoke zmogljivosti, lahko omejimo z dobro pripravo knjižnic spojin. Kemijske knjižnice spojin lahko sestavlja več milijonov spojin, ki imajo želene lastnosti za nadaljnje testiranje. Vsaki kemikaliji lahko določimo lastnosti, kot so kemijska struktura, čistost, količina in fizikalno-kemijske lastnosti spojine in jih shranimo v nekakšni zbirki podatkov. Našo knjižnico, ki vsebuje 887 spojin, smo pripravili s pomočjo virtualnega filtriranja v računalniškem programu KNIME iz kemikalij, ki jih že imamo na naši fakulteti. Cilj magistrske naloge je bil razvoj in optimizacija dveh metod visoke zmogljivosti za določanje topnosti in porazdelitvenega koeficienta, ki bi ju lahko uporabili na pripravljeni kemijski knjižnici. Za določanje topnosti smo uporabili metodo merjenja absorbance na mikrotitrski plošči in metodo HPLC. Na mikrotitrskih ploščah smo določali kinetično in termodinamsko topnost, ter preverili termodinamsko metodo še s pomočjo HPLC metode na reverznofazni C18 koloni. Za določanje porazdelitvenega koeficienta smo uporabili izokratsko metodo HPLC. Preizkušali smo tri različne kolone, različne mobilne faze in deleže organskega modifikatorja. V drugem delu smo metodi uporabili za določanje topnosti in porazdelitvenega koeficienta 219 modelnim spojinam iz kemijske knjižnice, ki smo jo pripravili z izbranimi filtri v računalniškem programu KNIME. Metodo merjenja termodinamske topnosti smo sicer razvili, a žal ni dala dobrih rezultatov, saj so le ti slabo ponovljivi in imajo slabo korelacijo z rezultati HPLC metode. Pravtako nismo bili uspešni z metodo za določanje porazdelitvenega koeficienta, saj le ta ni primerna za uporabo na takšnem številu spojin. Za nadaljnje delo predlagamo še dodaten razvoj in optimizacijo HPLC metode za določanje logP. The problem of false positives and hits with unfavourable properties obtained in high-throughput screening can be limited by good preparation of compound libraries. Chemical libraries can consist of up to a few million compounds that have the desired properties for further testing. Each compound can be characterized by properties such as chemical structure, purity, quantity and physicochemical properties of the compound. Our library, which contains 887 compounds, was prepared using virtual filtering in KNIME software from the chemicals on our faculty. The master's thesis aims to develop and optimize high-throughput methods for determining the solubility and partition coefficient that could be applied to our prepared chemical library. The microplate absorbance measurement method and the HPLC method were used to determine the solubility. We determined kinetic and thermodynamic solubility on microplates and then validated the thermodynamic method using the HPLC method on a reversed-phase C18 column. We used the isocratic HPLC method to determine the partition coefficient. We tested three different columns, different mobile phases and organic modifier proportions. Furthermore, these methods were used to determine the solubility and partition coefficient of 219 model compounds from the chemical library, which was prepared with selected filters in the KNIME software. The method of measuring thermodynamic solubility was developed but unfortunately it did not give good results, as these are poorly reproducible and had a poor correlation with the results of HPLC methods. The method for determining the partition coefficient was not suitable for use on such scale. The Master’s thesis outcomes represent starting points for further development and optimization of the logP HPLC method.

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.