Razvoj in validacija HPLC metode z uporabo pristopa »Analytical Quality by Design« (AqbD) : magistrsko delo

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Title:	Razvoj in validacija HPLC metode z uporabo pristopa »Analytical Quality by Design« (AqbD) : magistrsko delo
Authors:	ID Pavlovič, Marko (Author) ID Novak, Zoran (Mentor) More about this mentor... ID Perva, Amra (Comentor)
Files:	MAG_Pavlovic_Marko_2025.pdf (7,90 MB) MD5: 27DC42FB8ADA341B2E7B5199A5CB0089
Language:	Slovenian
Work type:	Master's thesis/paper
Typology:	2.09 - Master's Thesis
Organization:	FKKT - Faculty of Chemistry and Chemical Engineering
Abstract:	Magistrsko delo se osredotoča na razvoj in validacijo HPLC metode za določanje sorodnih substanc v raztopini klorheksidin diglukonata z uporabo pristopa Analytical Quality by Design (AQbD). Izhodišče raziskave je bila uradna metoda Evropske farmakopeje, ki uporablja kolono Luna C18 (25 cm × 4,6 mm, 5 μm) in gradientno ločbo s trifluoroocetno kislino v vodi in acetonitrilu. Metoda se je v praksi izkazala za neustrezno, saj ni zagotovila zadovoljive ločljivosti med nečistotama B in N ter O in I. Poleg tega kaže občutljivost na serijske razlike kolon, z sorazmerno dolgim časom kromatografije. Kot nadaljnji korak smo preučili revidirano različico metode, ki temelji na koloni Kinetex C18 (5 μm) in spremenjenem gradientu. Ta je izboljšala ločitev kritičnih parov nečistot, vendar podaljšala že sicer predolg čas analize. Za optimmizacijo revidirane metode smo uporabili programsko opremo LabSolutions MD (Shimadzu), ki temelji na načelih AQbD. Z načrtovanjem eksperimentov (DoE) in simulacijami kromatogramov smo sistematično analizirali vpliv pretoka in temperature kolone na ločljivost. S pomočjo orodij programa kot sta: analiza variance ANOVA ter matematična analiza modela, smo lahko časovno učinkovito in kvalitetno interpretirali rezultate eksperimentov. Podatke smo statistično obdelali ter opredelili robustno območje metode. Rezultati so pokazali, da kombinacija izbranih pogojev (pretok 1,5 mL/min, temperatura 25 °C) omogoča več 30-odstotno skrajšanje časa kromatografije glede na revidirano metodo, ob ohranjeni ustrezni ločljivosti vseh vrhov. Delna validacija je potrdila ponovljivost sistema in metode, linearno odzivnost, specifičnost ter robustnost. Magistrsko delo potrjuje, da kombinacija AQbD pristopa z uporabo programske opreme LabSolutions MD predstavlja zanesljivo in učinkovito orodje za razvoj HPLC metod.
Keywords:	Razvoj metode, AQbD, Klorheksidin diglukonat, LabSolutions MD
Place of publishing:	Maribor
Place of performance:	Maribor
Publisher:	[M. Pavlović]
Year of publishing:	2025
Number of pages:	1 spletni vir (1 datoteka PDF (XIV, 90 str.))
PID:	20.500.12556/DKUM-94508
UDC:	543.544.5:547.562(043.2)
COBISS.SI-ID:	288694787
Publication date in DKUM:	11.09.2025
Views:	181
Downloads:	50
Metadata:
Categories:	KTFMB - FKKT
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Licences

License:	CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Link:	http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:	The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:	19.08.2025

Secondary language

Language:	English
Title:	Analytical quality by design (AqbD) approach for an HPLC method development and validation
Abstract:	The master's thesis focuses on the development and validation of an HPLC method for the determination of related substances in chlorhexidine digluconate solution using the Analytical Quality by Design (AQbD) approach. The starting point of the research was the official method of the European Pharmacopoeia, which employs a Luna C18 column (25 cm × 4.6 mm, 5 μm) and gradient separation with trifluoroacetic acid in water and acetonitrile. In practice, the method proved to be inadequate, as it did not provide satisfactory resolution between impurities B and N, as well as O and I. In addition, it showed sensitivity to column batch differences and required a relatively long chromatographic run time. As a next step, we examined a revised version of the method, which is based on a Kinetex C18 column (5 μm) and a modified gradient. This improved the separation of critical impurity pairs but further prolonged the already lengthy analysis time. To optimize the revised method, we used the LabSolutions MD software (Shimadzu), which is based on AQbD principles. By applying design of experiments (DoE) and chromatogram simulations, we systematically analyzed the influence of flow rate and column temperature on resolution. With the help of software tools such as analysis of variance (ANOVA) and mathematical model evaluation, we were able to interpret the experimental results in a time-efficient and reliable manner. The data were statistically processed, and the robust method region was defined. The results showed that the combination of selected conditions (flow rate 1.5 mL/min, column temperature 25 °C) allows more than a 30% reduction in chromatographic run time compared to the revised method, while maintaining adequate resolution of all peaks. Partial validation confirmed system and method repeatability, linearity, specificity, and robustness. The master's thesis confirms that the combination of the AQbD approach with the use of LabSolutions MD software represents a reliable and effective tool for the development of HPLC methods.
Keywords:	Method development, AQbD, Chlorhexidine digluconate, LabSolutions MD

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