Razvoj metode za določevanje urolitina A v realnih vzorcih

Kržan, Pia (author), Pompe, Matevž (mentor)

Urolitini so metaboliti elagitaninov, kateri nastanejo s pomočjo črevesnih bakterij. Veliko pozornosti so pritegnili zaradi potencialnih antioksidativnih, protivnetnih, protirakavih učinkov in delovanja proti nevrodegenerativnim boleznim. Poteka še veliko raziskav o samih spojinah in njihovih zdravilnih učinkih. Pomemben je tudi razvoj metod, s katerimi se lahko urolitine določa v različnih bioloških vzorcih. V tej nalogi sem se osredotočila na kvalitativno in kvantitativno določitev urolitina A v vzorcih prašičjega blata. Tovrstni vzorci so najlažje dostopni in lahko povedo veliko o metabolizmu. Razvita je bila metoda za pripravo vzorca in analizo na sistemu revezno fazne tekočinske kromatografije visoke ločljivosti z UV-Vis detekcijo. Preizkušenih je bilo več načinov in topil za ekstrakcijo vzorca. Razvita je bila ekstrakcija tekoče-tekoče z raztopino vzorca in etil acetatom, s katero se vzorec skoncentrira, vendar je pri postopku veliko izgub. Za najbolj optimalno se je izkazala enofazna ekstrakcija z mešanico topil metanola in vode v razmerju 80:20 z dodatkom kisline. Vzorci blata so kompleksni in opažena je bila velika raznolikost v vsebnosti motečih komponent, ki so v nekaterih primerih vplivali na določevanje urolitina A v vzorcu. Najboljša ločba je bila dosežena s stacionarno fazo z vezanimi fenil-heksil skupinami in gradientnim profilom mobilnih faz 0,1 % raztopine H3PO4 v vodi ter acetonitrila. Uporabljena optimalna valovna dolžina je bila 240 nm. Določena je bila linearnost, ponovljivost metode, LOD, LOQ in vpliv matrice. Metoda je primerna za kvalitativno določevanje, kvantitativno določevanje pa je v določenih primerih moteno zaradi prisotnost motečih komponent. Preizkušena je bila topnost urolitina A v topilih metanol, mešanici metanola in vode v razmerju 80:20, vodi in etil acetatu. Najbolj topen je v metanolu s topnostjo 0,737 g/L in najslabše v vodi s topnostjo 0,170 g/L. Preverjena je bila stabilnost urolitina A v topilih DMSO, metanolu in mešanici metanola in vode v razmerju 80:20 pri koncentracijah osnovne in delovne raztopine ter pri različnih pogojih: v zamrzovalniku pri -20 °C, v hladilniku pri 4 °C, v temi pri sobni temperaturi in na svetlobi pri sobni temperaturi. Stabilnost je upadala le v primeru raztopine, ki je bila izpostavljena svetlobi v topilu DMSO.

• Language: Slovenian
• Keywords: HPLC, urolitin A, biološki vzorci
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-141333
• COBISS.SI-ID: 132100355
• Publication date in RUL: 28.09.2022

Secondary language

• Language: English
• Title: Development of a method for the determination of urolithin A in real samples

Abstract

Urolithins are metabolites of ellagitannins produced by intestinal bacteria. They have attracted a lot of attention due to their potential antioxidant, anti-inflammatory, anti-cancer effects and their action against neurodegenerative diseases. There is still a lot of research going on about the compounds themselves and their health effects. The development of a method by which urolithin can be determined in various biological samples is also important. In this master thesis, I focused on the qualitative and quantitative determination of urolithin A in pig feces samples. These types of samples are the most easily accessible and can tell a lot about the metabolome. A method for sample preparation and analysis on a reverse-phase high-performance liquid chromatography system with UV-Vis detection was developed. Several methods and solvents to extract the sample were tested. A liquid-liquid extraction with a solution of the sample and ethyl acetate has been developed to concentrate the sample, but there are many losses in the process. Single-phase extraction with a solvent mixture of methanol and water in a ratio of 80:20 with the addition of acid proved to be the optimal. Stool samples are so complex and a great diversity in the content of disturbing components was observed, which in some cases affected the determination of urolithin A in the sample. The best separation was achieved with a stationary phase with attached phenyl-hexyl groups and a mobile phase gradient profile of 0.1 % solution of H3PO4 in water and acetonitrile. The optimal wavelength used was 240 nm. Linearity, method reproducibility, LOD, LOQ and matrix effect were determined. The method is suitable for qualitative determination, but quantitative determination is disturbed in certain cases due to the presence of interfering components. The solubility of urolithin A was tested in the solvent methanol, an 80:20 mixture of methanol and water, water and ethyl acetate. It is best soluble in methanol with a solubility of 0.737 g/L and the worst in water with a solubility of 0.170 g/L. The stability of urolithin A was checked in the solvents DMSO, methanol and a mixture of methanol and water in a ratio of 80:20 at the concentrations of the stock and working solution and under different conditions: in a freezer at -20 °C, in a refrigerator at 4 °C, in the dark at room temperature and in the light at room temperature. The stability dropped only in the case of the solution exposed to light in the DMSO solvent.

• Keywords: HPLC, urolithin A, biological samples