Razvoj robustne masno spektrometrične metode za določanje metabolne stabilnosti spojin vodnic

Bukovac, Nina (author), Ilaš, Janez (mentor)

Kromatografija je ena izmed najpogosteje uporabljanih analitskih tehnik za ločevanje in identifikacijo spojin. Poznamo različne kromatografske tehnike, ki se razlikujejo na podlagi različnih mobilnih in stacionarnih faz. Najbolj razširjena med njimi je tekočinska kromatografija, ki temelji na tekoči mobilni fazi in trdni ali tekoči stacionarni fazi med katerima se porazdeljujejo analiti. Kromatografija je zaradi svoje univerzalnosti in vsestranske uporabe v farmacevtski industriji nepogrešljiva. Pomembno vlogo v razvoju, raziskavah in proizvodnji zdravil ima sam razvoj analiznih metod, ki je precej kompleksen, saj zahteva upoštevanje velikega števila faktorjev in izkušenj. V prvem in glavnem delu magistrske naloge smo razvili analizno metodo s tekočinsko kromatografijo visoke ločljivosti sklopljeno z masnim spektrometrom, s katero je možno ločiti čim večje število različnih spojin vodnic v čim krajšem času. Ko smo uspeli razviti ustrezno analizno metodo z optimalnimi kromatografskimi pogoji, smo v nadaljevanju naloge preverili metabolno stabilnost naših spojin. In sicer tako, da smo jih testirali ob času 0 in po 30 minutah inkubiranja v prisotnosti S9 frakcije metabolnih encimov. Spojine, ki so po 30 minutah izražale podvrženost metabolizmu (površina vrha se jim je glede na začetno vrednost zmanjšala za vsaj 10 %) smo nato testirali v časovnih točkah 0, 15, 30, 60 in 120 minut. Preučevali smo 17 spojin, med katerimi se jih je 12 pri testiranju ob časih 0 in 30 minut izkazalo za metabolno stabilne, z razpolovnim časom večjim od 5-ih ur. 5 spojin pa je z S9 encimsko frakcijo izražalo podvrženost metabolizmu. Posameznim spojinam smo na podlagi spremembe površine pod krivuljo v UV kromatogramu določili razpolovne čase. V zadnjem delu naloge smo nastalim metabolitom poizkušali predlagati možno strukturo. Strukture nastalih metabolitov smo predlagali na podlagi določene molske mase spojine. Pri določenih spojinah metaboliti zaradi nizkih odzivov v UV kromatogramu niso bili vidni in smo jih lahko spremljali samo z masnim detektorjem.

• Language: Slovenian
• Keywords: tekočinska kromatografija visoke ločljivosti, masna spektrometrija, razvoj metode, metabolna stabilnost
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-121113
• Publication date in RUL: 30.09.2020

Secondary language

• Language: English
• Title: Development of robust mass spectrometric method for determination of lead compounds metabolic stability

Abstract

Chromatography is one of the most commonly used analytical techniques for the separation and identification of compounds. We know different chromatographic techniques that differ based on different mobile and stationary phases. The most common of these is liquid chromatography which is based on the liquid mobile phase and the solid or liquid stationary phase between which the analytes are partitioned. Chromatography is indispensable due to its universality and versatility in the pharmaceutical industry. An important role in the development, research, and production of medicines is played by the development of analytical methods which is quite complex as it requires consideration of a large number of factors and experience. In first and the main part of our project we developed an analytical method with high performance liquid chromatography coupled with mass spectrometer which is able to separate as many different conductor compounds as possible in the shortest possible time. Once we were able to develop an appropriate analytical method with optimal chromatographic conditions, we further verified the metabolic stability of our compounds by testing them at time 0 and after 30 minutes of incubation. Compounds that expressed metabolic instability after 30 minutes (their peak surface area decreased by at least 10 % compared to the initial one) were then tested at 0, 15, 30, 60, and 120 minute time points. We studied 17 compounds, of which 12 proved to be metabolically stable when tested at times 0 and 30 minutes, with a half-life greater than 5 hours. Other 5 compounds expressed their susceptibility to metabolism with the S9 enzyme fraction. The half-lives of individual compounds were determined in a UV chromatogram based on the change in area under the curve. In the last part of the task, we tried to suggest a possible structure to the resulting metabolites. The structures of the resulting metabolites were proposed based on the determined molecular mass. For certain compounds, the metabolites were not visible in the UV chromatogram due to low responses and could only be monitored by mass detector.

• Keywords: high performance liquid chromatography, mass spectrometry, method development, metabolic stability