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Kolagen je najbolj zastopan protein v človeškem telesu in ima pomembno vlogo pri ohranjanju strukture različnih tkiv. Zaradi njegovih učinkov na kožo, kosti, sklepe in mišice najdemo na trgu vedno več prehranskih dopolnil s kolagenom. Kolagen ima kompleksno strukturo, zato za njegovo karakterizacijo uporabljamo več različnih analiznih metod. Glavni namen magistrske naloge je bila optimizacija enostavnih spektroskopskih metod za vrednotenje vsebnosti kolagena v prehranskih dopolnilih. Osredotočili smo se na UV-VIS in fluorimetrično metodo. Z metodo UV-VIS vsebnost kolagena določamo posredno preko aminokisline 4-hidroksiprolin, ki nastane po kemijski hidrolizi kolagena. Fluorimetrična metoda pa temelji na encimski razgradnji kolagena do tripeptidov, ki jih kompleksiramo do fluorescirajočega produkta. Pri optimizaciji obeh metod smo spreminjali pogoje hidrolize kolagena in derivatizacijske reakcije ter izbirali tiste, s katerimi smo poenostavili postopek priprave vzorcev in hkrati dosegli čim večjo občutljivost in ustrezno ponovljivost metode. Metodi smo ovrednotili v skladu s smernicami ICH. Obe metodi sta ustrezali postavljenim kriterijem sprejemljivosti. Z vidika linearnosti, analiznega območja, točnosti, natančnosti in nižje meje določitve je boljša fluorimetrična metoda. Ovrednotili smo tudi stabilnost vzorcev kolagena in 4-hidroksiprolina in ugotovili, da moramo pri fluorimetrični metodi vzorce po dodatku vseh reagentov pomeriti v roku 2 ur od priprave, pri metodi UV-VIS pa v roku 4 ur od priprave. Obe metodi smo uporabili za ugotavljanje vsebnosti kolagena v dvanajstih prehranskih dopolnilih. Ugotovljene vsebnosti smo primerjali z navedenimi in ugotovili, da so bile v območju med 78 in 130 % navedene vsebnosti, kar je v skladu s pričakovanji glede na ohlapno regulativo za prehranska dopolnila. Z obema metodama smo dobili primerljive rezultate za vsebnost kolagena, a nobena v celoti ne ustreza postavljenim kriterijem sprejemljivosti metoda UV-VIS je zaradi večjega sipanja rezultatov bolj uporabna kot semi-kvantitativna metoda, fluorimetrična metoda pa ima velik potencial za vrednotenje vsebnosti kolagena v prehranskih dopolnilih. Razvita analizna metodologija je ustrezna za predvideni namen uporabe, a je smiselna nadaljnja optimizacija obeh uporabljenih metod in souporaba komplementarnih, bolj selektivnih kromatografskih metod. Collagen is the most abundant protein in the human body and provides structural integrity to various tissues. Due to its effect on skin, bones, joints, and muscles, there is a growing interest in collagen supplements. The structure of collagen is rather complex, and therefore various analytical methods are used to characterize it. The main goal of our work was to optimize simple spectroscopic methods used for the evaluation of collagen content in food supplements. Our focus was on the UV-VIS and fluorometric methods. Using the UV-VIS method, the collagen content is determined indirectly – via the amino acid 4-hydroxyproline, which is a product of the chemical hydrolysis of collagen. The fluorometric method is based on the enzymatic degradation of collagen to tripeptides, which are complexed into a fluorescent product. During the optimization of both methods, the conditions of collagen hydrolysis and derivatization reactions were modified. The conditions that result in simplification of the sample preparation procedure and improvement in sensitivity and repeatability of the method were selected. The methods were evaluated according to the ICH guidelines. Both methods met the acceptance criteria, but the fluorometric method proved better in terms of linearity, analytical range, accuracy, precision, and limit of quantification. The stability of collagen and 4-hydroxyproline in the samples was also evaluated. When using the fluorometric method and the UV-VIS method, the samples must be analyzed within two and four hours, respectively, after the addition of all reagents. The validated methods were used to evaluate collagen content in twelve food supplements. The determined contents were compared with the declared ones and were found to be between 78 and 130 % of the declared content, which is in line with the expectations, considering food supplements legislation. Similar collagen contents in food supplements were determined by both methods. The UV-VIS method is useful as a semi-quantitative method due to the scattering of the results while the fluorometric method has great potential as a method for the quantitative evaluation of collagen content in food supplements. The developed analytical methodology is suitable for the intended use, but it is reasonable to further optimize both methods used and to include complementary, more selective chromatographic methods. |
