Validacija metode za določanje ogljikovih hidratov s fluorescenčno detekcijo z uporabo pristopa "Quality by Design"

Kogovšek, Jan (author), Kočar, Drago (mentor)

Glikani imajo ključno vlogo v različnih bioloških procesih, vključno s celičnim signaliziranjem, imunskim odzivom in funkcionalnostjo proteinov. Zaradi njihove strukturne raznolikosti in kompleksnosti je njihova analiza zahtevna, vendar bistvena za razumevanje njihovega funkcionalnega pomena v biologiji. Ker naravno nimajo lastnosti oddajanja svetlobe, je za njihovo odkrivanje in analizo potrebna derivatizacija s fluorofori. Fluorescenčno zaznavanje ima pri analizi glikanov več prednosti, vključno z večjo občutljivostjo in možnostjo zaznavanja nizkih koncentracij analitov. V študiji sem razvil analitično metodo HPLC za glikane z zaznavanjem fluorescence, ki omogoča učinkovito karakterizacijo in kvantifikacijo teh biomolekul. Poleg tega sem želel uporabiti načela QbD (Quality by Design) za zagotavljanje robustnosti in zanesljivosti pri razvoju analitske metod. Uporaba načel QbD pri razvoju analitskih metod omogoča sistematičen in celovit pristop za doseganje želene analitske učinkovitosti. Okvir QbD omogoča aktiven pristop k razvoju metod, ki vključuje oceno tveganja, načrtovanje poskusov in večdimenzionalno analizo. Ta pristop zagotavlja robustnost metode, zmanjšuje variabilnost in izboljšuje razumevanje metode, kar na koncu vodi do zanesljive in ponovljive analize glikanov. V laboratoriju sem uporabili metode ekstrakcije na trdno fazo (SPE) za čiščenje vzorcev glikanov, odstranitev motečih snovi in zmanjšanje učinkov matriksa. S skrbno izbiro ustreznih sorbentov SPE in optimizacijo pogojev ekstrakcije sem dosegel učinkovito odstranitev odvečnega fluorescenčnega označevalca, uporabljenega za zaznavo. Ta korak je ključnega pomena, saj lahko visoko fluorescenčno ozadje vpliva na občutljivost in natančnost kvantifikacije glikanov. Poleg tega sem v celotnem postopku razvoja metode izvajal načela QbD, kar je omogočilo temeljito opredelitev kritičnih parametrov metode, oceno tveganja ter opredelitev kritičnih parametrov procesa (CPP) in kritičnih lastnosti kakovosti (CQA).

• Language: Slovenian
• Keywords: HPLC, ogljikovi hidrati, fluorescenčna detekcija, Quality by Design, ekstrakcija na trdo fazo.
• Work type: Bachelor thesis/paper
• Typology: 2.11 - Undergraduate Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2023
• PID: 20.500.12556/RUL-149317
• COBISS.SI-ID: 167964931
• Publication date in RUL: 06.09.2023

Secondary language

• Language: English
• Title: Validation of a method for carbohydrate determination with fluorescence detection using Quality by Design approach

Abstract

Glycans play a crucial role in various biological processes, including cell signalling, immune response, and protein functionality. Their structural diversity and complexity make their analysis challenging, but essential for understanding their functional importance in biology. Since they naturally lack light-emitting properties, derivatization with fluorophores is required to enable their detection and analysis. Fluorescence detection offers several advantages in glycan analysis, including higher sensitivity and the ability to detect low concentrations of analytes. In this study, I aim to develop an analytical HPLC method for glycans with fluorescence detection that allows efficient characterization and quantification of these biomolecules. Furthermore, I apply the principles of Quality by Design (QbD) to ensure robustness and reliability in the development of analytical methods. Applying the QbD principles to the development of analytical methods enables a systematic and comprehensive approach to achieve the desired analytical performance. The QbD framework enables a proactive approach to method development that includes risk assessment, experimental design, and multivariate analysis. This approach ensures method robustness, minimizes variability and improves method understanding, ultimately leading to reliable and reproducible glycan analysis. In the laboratory, I used solid-phase extraction (SPE) methods to purify glycan samples, eliminate interfering substances, and reduce matrix effects. By carefully selecting appropriate SPE sorbents and optimizing extraction conditions, I achieved efficient removal of excess fluorescent markers used for detection. This step is crucial as a high fluorescence background can affect the sensitivity and accuracy of glycan quantification. In addition, I implemented QbD principles throughout the method development process, which enabled thorough characterization of critical method parameters, risk assessment and identification of critical process parameters (CPPs) and critical quality attributes (CQAs).

• Keywords: HPLC, glycans, fluorescence detection, Quality by Design, solid phase extraction.