Vrednotenje uporabnosti linearnega pH-gradienta za kromatografsko določitev konstante disociacije

Murn, Neža (author), Žakelj, Simon (mentor), Trontelj, Jurij (co-mentor)

Kislinska konstanta disociacije, pKa, je s svojim vplivom na farmakokinetične lastnosti učinkovin ena najpomembnejših fizikalno-kemijskih lastnosti. Zaradi številnih vplivov je poznavanje pKa vrednosti učinkovin že zgodaj v farmacevtskem razvoju bistvenega pomena. Uporaba konvencionalnih metod za njeno določanje, kot sta potenciometrična titracija in spektrofotometrija, je z uveljavitvijo sodobnejših pristopov razvoja novih zdravilnih učinkovin v farmacevtski industriji postala omejena. Potreba po razvoju novih, visokozmogljivostnih metod za hitro in učinkovito določanje pKa vrednosti je zato pomembna tema raziskovanj na področju farmacevtske analitike. Kot ustrezna visokozmogljivostna metoda za določitev konstante disociacije se je v nekaterih raziskavah izkazala reverzno-fazna HPLC metoda z linearnim pH gradientom, saj omogoča analizo majhnih količin analita, tudi ko je topnost slaba, kljub temu pa njena uporaba ni široko razširjena, zato smo se v okviru raziskovalnega dela odločili za proučitev predlagane metode z vidika njenih slabosti in prednosti. Na naboru 40 učinkovin različnih fizikalno-kemijskih lastnosti smo preverjali uporabnost opisanih postopkov metode. Za vsako od učinkovin smo izvedli dve analizi z gradientom metanola, na podlagi katerega smo določili nadaljnje pogoje kromatografske analize in jih izvedli sprva na HPLC sistemu z binarno črpalko ter kasneje s kvarterno črpalko. S končnimi nastavitvami smo prišli do uporabnih rezultatov za 31 učinkovin in dobljene pKa vrednosti primerjali z referenčnimi iz literature, ki so izkazali relativno dobro korelacijo. Ugotovili smo, da poleg ustrezne strojne opreme kritično točko v uporabi metode predstavljata ustreznost kemizma uporabljene kolone v povezavi z linearnostjo pH gradienta, ki ga zagotavlja primerna sestava pufrne raztopine. Ugotovljena je bila tudi omejena uporabnost metode na polarnejših analitih. Kot pomemben doprinos k obstoječi metodi smo v našem delu podrobneje preverili linearnost pH gradienta z izbrano sestavo pufra in uvedli dodaten postopek linearizacije pH gradienta. Na podlagi naših izkušenj smo prišli do zaključka, da ima kromatografsko določanje pKa vrednosti velik potencial, vendar bi bilo za avtomatizirano uporabo metode treba celoten postopek optimizirati z vidika omejujočih dejavnikov s katerimi smo se srečali pri našem delu (npr. kemizem stacionarne faze).

• Language: Slovenian
• Keywords: kislinska konstanta disociacije, pKa vrednost, linearen pH gradient, reverzno-fazni HPLC sistem, HPLC
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-144871
• Publication date in RUL: 18.03.2023

Secondary language

• Language: English
• Title: Evaluation of linear pH gradient applicability for chromatographic determination of dissociation constant

Abstract

Dissociation constant, pKa value, is a significant physicochemical characteristic of an active pharmaceutical substance due to its important impact on different pharmacocinetic properties. Therefore the knowledge of this value of drug candidates and interest in the development of new methods for its determination, especially in early stages of drug discovery, is of great importance, since the traditional techniques, such as potenciometry and spectrophotometry, cannot meet the standards of the modern approaches to the search for new drugs, which demands the development of high-throughput methods for fast and effective determination of pKa value. Reverse-phase high-performance liquid chromatography with linear pH gradient was presented as a suitable high-thorughput method for pKa determination, since it is appropriate for analytes in small amounts and with poor solubility. Nevertheless we did not find this method to be widely used, which is why we decided to study it in terms of its advantages and disadvantages. The evaluation of pH gradient RF HPLC applicability for pKa determination was studied on series of 40 analytes with different properties. We first carried out two methanol gradient runs at two different gradient times for each analyte, from which a proper methanol content in mobile phase was evaluated for further chromatographic analysis which were first carried out on HPLC sistem with binary and later with quaternary pump. Finally we managed to calculate and compare pKa values for 31 analytes with reference values. The results showed quite satisfying correlation. We learned that next to proper choice of equipment the most important part of the method represents the column chemistry and a linear pH gradient, which is provided by appropriate buffer solution. We demonstrated that the method cannot be properly used with hydrophilic compounds. As an important contribution to the existing method we introduced an additional procedure of buffer linearization for pH gradient. Based on our experience we can confirm great potential of RF HPLC method for pKa determination, although some optimization of the method would be needed for automated use (such as the choice of stationary phase used for HPLC analysis).

• Keywords: acid dissociation constant, pKa value, linear pH gradient, reverse-phase high-performance liquid chromatography, HPLC