Vrednotenje orodja za razvoj tekočinsko-kromatografskih metod in silico na primeru izbranih učinkovin

Janežič, Blaž (author), Mravljak, Janez (mentor)

Razvoj analitskih metod je kontinuiran proces, ki spremlja metodo skozi njen življenjski cikel, od začetnega razvoja do implementacije v kontrolo kakovosti in prenosov med laboratoriji. Tradicionalni pristop naključnega preizkušanja je dolgotrajen in omejen pri odkrivanju interakcij med parametri. Alternativo predstavlja pristop razvoja z vgrajeno kakovostjo, ki uporablja statistične metode in načrtovanje eksperimentov ter se osredotoča na sistemski razvoj, kjer kakovost načrtujemo in razumemo že v samem začetku procesa razvoja. Smernica mednarodnega sveta za harmonizacijo tehničnih zahtev za zdravila za uporabo v humani medicini Q14 iz leta 2024 uvaja koncept razvoja z vgrajeno kakovostjo tudi na področju analitskih metod. Razvoj metod in silico temelji na matematičnih modelih za napovedovanje vpliva kromatografskih pogojev. V magistrski nalogi sta bili razviti in preizkušeni analizni metodi tekočinske kromatografije visoke ločljivosti za analizo ibuprofena in ketoprofena ter za referenčni pripravek z lidokainom, 2,6-ksilidinom, metilparabenom in propilparabenom. V nalogi je prikazan sistematičen razvoj metod, ki vključuje ovrednotenje fizikalno-kemijskih lastnosti analitov, postavitev zahtev ustreznosti metode, izbiro začetnih pogojev, izvedbo izvidniškega gradienta in optimizacijo z različnimi mobilnimi fazami in tipi stacionarnih faz. V okviru magistrskega dela smo za ovrednotenje orodja Practical HPLC Simulator preverili hipoteze ujemanja retencijskega časa, učinkovitosti kolone in faktorja ločbe med napovedmi in silico in eksperimentalno določenimi rezultati na sistemu HPLC. Simulator je sicer točno napovedal nekatere parametre, kot sta faktor ločbe in simetrija vrhov, vendar je retencijski čas pogosto napačno ocenjen, pri čemer je stopnja odstopanja odvisna od posameznega analita. Glede na pridobljene rezultate smo zaključili, da je orodje Practical HPLC Simulator uporabno za hitro testiranje sprememb parametrov metod in silico, vendar njegova omejena točnost onemogoča uporabo kot primarnega orodja za razvoj metod. Ugotovili smo, da ima potencial kot pripomoček v začetnih fazah razvoja preprostih metod in kot izobraževalno orodje. Nadaljnje raziskave bi morale vključevati širši spekter molekul in razlago odstopanj med napovedmi in dejanskimi meritvami.

• Language: Slovenian
• Keywords: tekočinska kromatografija, Practical HPLC Simulator, razvoj metod, simulator, ibuprofen, ketoprofen, lidokain
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2025
• PID: 20.500.12556/RUL-167692
• Publication date in RUL: 07.03.2025

Secondary language

• Language: English
• Title: Evaluation of an in silico tool for the development of liquid chromatographic methods on the example of selected active compounds

Abstract

The development of analytical methods is a continuous process that accompanies the method throughout its lifecycle, from initial development to implementation in quality control and transfers between laboratories. The traditional trial-and-error approach is time-consuming and limited in detecting interactions between parameters. An alternative is the quality by design approach, which utilizes statistical methods and experimental design experiments and focuses on systematic development, where quality is planned and understood at the beginning of the development process. The 2024 International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use Q14 guideline introduced quality by design principles to analytical method development. In silico method development relies on mathematical models to predict the effects of chromatographic conditions. In the master's thesis, an HPLC method was developed and tested for the analysis of ibuprofen and ketoprofen, as well as an HPLC method for a formulation containing lidocaine, 2,6-xylidine, methyl paraben and propyl paraben. In this thesis, a systematic method development is presented, including the evaluation of the physicochemical properties of the analytes, setting the requirements for method suitability, selecting initial conditions, performing a scouting gradient, and optimizing the method. Within the master's thesis, we evaluated the Practical HPLC Simulator tool by examining the hypotheses of matching retention time, column efficiency, and separation factor between in silico predictions and experimentally determined results on the system. The simulator accurately predicted some parameters, such as the separation factor and peak symmetry, but often incorrectly estimated the retention time, with the degree of deviation depending on the individual analyte. According to the obtained results the simulator is useful for rapidly testing method modifications however, its limited accuracy restricts its use as a primary tool. We concluded that the simulator has potential as a tool in the initial stages of developing simple methods and as an educational aid. Further research should include a broader range of molecules and investigate the discrepancies between predictions and actual measurements.

• Keywords: liquid chromatography, Practical HPLC Simulator, method development, simulator, ibuprofen, ketoprofen, lidocaine