Razvoj kromatografskega pristopa za vrednotenje vsebnosti kolagena v prehranskih dopolnilih

Kurent, Ema (author), Roškar, Robert (mentor), Kržišnik, Nika (comentor)

Kolagen je najbolj zastopan protein pri ljudeh in živalih. Uživanje prehranskih dopolnil s kolagenom ima pozitivne učinke predvsem na kožo in sklepe. Ker ima kolagen kompleksno strukturo, za karakterizacijo uporabljamo več metod, kot so spektroskopske, kromatografske in druge. Namen magistrske naloge je bil razviti in optimizirati več enostavnih kromatografskih metod za vrednotenje vsebnosti kolagena v prehranskih dopolnilih. Osredotočili smo se na reverznofazno in izključitveno kromatografijo. Pri optimizaciji smo preizkusili različne kolone, mobilne faze, gradiente in pretoke. Najprimernejšo metodo smo izbrali iz vidika ločbe, oblike kromatografskih vrhov in časa analize enega vzorca. Izključitveno kromatografijo smo uporabljali zgolj za kvalitativno vrednotenje velikosti kolagenskih peptidov v prehranskih dopolnilih. Poleg tega smo razvili tudi štiri reverznofazne kromatografske metode, ki omogočajo kvantitativno določitev kolagena za različne priprave vzorcev. Vsebnost kolagena smo določali v intaktnih hidrolizatih kolagena in v encimsko hidroliziranem vzorcu, pri katerem smo določali vsebnost kolagena preko tripeptida Gly Pro Hyp. Hidroliziran vzorec smo tudi derivatizirali do fluorescenčnega produkta in zanj razvili kromatografsko metodo s fluorescenčno detekcijo. S četrto metodo smo vsebnost kolagena določali po bazični hidrolizi kolagena do aminokislin, pri čemer smo derivatizirali hidroksiprolin. Razvite reverznofazne kromatografske metode smo validirali v skladu s smernicami ICH. Vse metode so ustrezale postavljenim kriterijem sprejemljivosti. Razvite kromatografske metode smo primerjali s predhodno postavljenimi spektroskopskimi metodami. Kromatografske metode so bolj selektivne in nekatere omogočajo razlikovanje med ribjim in govejim kolagenom. Z vidika stabilnosti vzorcev, validacijskih parametrov in enostavnosti se je kot najboljša izkazala metoda, s katero določamo vsebnost kolagena v intaktnih hidrolizatih kolagena. Z razvitimi metodami smo analizirali trinajst prehranskih dopolnil in ugotovljene vsebnosti primerjali z navedenimi. Vsebnosti za večino kolagenskih hidrolizatov so bile znotranj intervala 80 in 115 %, kar je v skladu s pričakovanji, saj je regulativa za prehranska dopolnila manj stroga kot za zdravila. Z različnimi metodami smo dobili primerljive rezultate, metodologija pa ustreza predvidenemu namenu uporabe.

• Language: Slovenian
• Keywords: 4-hidroksiprolin, HPLC, kolagen, prehranska dopolnila, tripeptid
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2024
• PID: 20.500.12556/RUL-158748
• Publication date in RUL: 20.06.2024

Secondary language

• Language: English
• Title: Development of a chromatographic approach for evaluation of collagen content in food supplements

Abstract

Collagen is the most abundant protein in humans and animals. Taking food supplements with collagen has positive effects, especially on the skin and joints. Due to its complex structure, various techniques such as spectroscopic, chromatographic and others are used for characterization of collagen. The main objective of our work was to develop and optimize several simple chromatographic methods for the evaluation of collagen content in food supplements. We focused on reversed-phase and size-exclusion chromatography. Methods were optimized by testing different columns, mobile phases, gradients and flow rates. The final methods were selected based on resolution, shape of chromatography peaks and run time for a sample. Size-exclusion chromatography was used only for the qualitative evaluation of the size of collagen peptides in food supplements. In addition, four reversed-phase chromatographic methods for the quantitative determination of collagen content for various sample preparations were developed. The collagen content was determined in intact collagen hydrolysates and in the enzymatically hydrolysed sample, in which the collagen content was determined via the tripeptide Gly-Pro-Hyp. A chromatographic method with fluorescence detection was developed for the derivatized enzymatically hydrolysed sample. In the fourth method, the collagen content was determined by basic hydrolysis of collagen to amino acids, whereby hydroxyproline was derivatized. The selected reversed-phase methods were validated according to ICH guidelines. All four methods met the acceptance criteria. The developed chromatographic methods were compared with already established spectroscopic methods. Chromatographic methods are more selective, some also distinguish between marine and bovine collagen. The method for intact collagen hydrolysates proved to be the best in terms of sample stability, validation parameters and simplicity. Thirteen food supplements were analysed with the developed methods and the determined contents were compared with the declared contents. The contents for the most of collagen hydrolysates were between 80 and 115 %, which is in line with expectations, as the regulations for food supplements are less strict than those for medicines. Comparable results were obtained using different methods and the methodology is suitable for the intended use.

• Keywords: collagen, food supplements, HPLC, 4-hydroxyproline, tripeptide