Search

Vitamini so zelo aktualno raziskovalno področje zaradi številnih pozitivnih učinkov pri ohranjanju zdravja. V splošnem so vodotopni vitamini nestabilni, a je podatkov malo, še posebej o njihovi stabilnosti v trdnem stanju. Naš namen je bil pridobiti podatke o stabilnosti vodotopnih vitaminov v trdnem stanju, posamezno in v zmeseh. Glavni cilj magistrske naloge je bil razvoj enostavnega analiznega pristopa za spremljanje stabilnosti najpogostejših oblik vodotopnih vitaminov v trdnem stanju. V ta namen smo predhodno razvito stabilnostno indikativno metodo UHPLC-DAD za sočasno vrednotenje glavnih vodotopnih vitaminov (tiaminijev klorid (B1), riboflavin (B2), riboflavin natrijev fosfat (B2-f), nikotinamid (B3), kalcijev D-pantotenat (B5), piridoksinijev klorid (B6), D-biotin (B7), folna kislina (B9), cianokobalamin (B12), L-askorbinska kislina (C)) razširili za vrednotenje dveh dodatnih vitamerov B3 in B9 (nikotinska kislina in 5-metil tetrahidrofolna kislina). Z namenom skrajšanja časa in poenostavitve analiznega postopka smo razvili in optimizirali kratke stabilnostno indikativne metode za analizo posameznih vodotopnih vitaminov. Vse metode smo ovrednotili v skladu s smernicami ICH in jih uporabili pri vrednotenju stabilnosti vodotopnih vitaminov v trdnem stanju, posamezno in v zmeseh. Stabilnostne študije smo izvedli v klimatskih komorah pri različnih stresnih pogojih – povišani temperaturi, relativni vlažnosti, svetlobi. Optimizirali smo pripravo vzorcev in prilagajali eksperimentalne pogoje. Na osnovi rezultatov trimesečne študije stabilnosti posameznih vitaminov pri 60 °C in 75 % relativne vlažnosti smo potrdili ustrezno stabilnost (> 90 % začetne vsebnosti) za večino vitaminov in manjšo stabilnost (45 – 89 %) za vitamine B5, B12 in obe obliki B2. V enotedenski študiji izpostavitve posameznih vodotopnih vitaminov svetlobi smo ugotovili, da vsi vitamini razen vitaminov C in B12 ohranjajo svojo stabilnost (> 90 % začetne vsebnosti). Vrednotenje stabilnosti vodotopnih vitaminov v zmesi je bilo težavno zaradi nehomogenosti vzorcev. Smo pa ugotovili, da so vitamini v zmesi manj stabilni kot posamezno, kar so potrdile tudi organoleptične spremembe vzorcev (ožganost in smoljnatost). Na stabilnost vitaminov v zmesi je pomembno vplivala zlasti višja relativna vlažnost in njen sinergistično destabilizirajoči učinek s povišano temperaturo. Zaključimo lahko, da smo uspešno razvili analizni pristop za vrednotenje stabilnosti posameznih vodotopnih vitaminov v trdnem stanju. Za ustrezno ovrednotenje stabilnosti vitaminov v zmeseh pa bo potrebna še nadaljnja optimizacija pred-analiznih aktivnosti. Vitamins are a very topical field of research due to their many positive effects in maintaining health. In general, water-soluble vitamins are unstable, but little data is available, especially about the stability of water-soluble vitamins in the solid state. Our purpose was to obtain data on their stability in the solid state, individually and in mixtures. The main goal of the master's thesis was to develop a simple analytical approach to monitor the stability of the most common forms of water-soluble vitamins in the solid state. For this purpose we expanded previously developed stability indicative UHPLC-DAD method for the simultaneous evaluation of the main water-soluble vitamins (thiamine chloride (B1), riboflavin (B2), riboflavin sodium phosphate (B2-f), nicotinamide (B3), calcium D-pantothenate (B5), pyridoxine hydrochloride (B6), D-biotin (B7), folic acid (B9), cyanocobalamin (B12), L-ascorbic acid (C)) for evaluation of two additional vitamers B3 and B9 (nicotinic acid and 5-methyl tetrahydrofolic acid). In order to shorten the time and simplify the analytical procedure, we have developed and optimized fast stability-indicative methods for the analysis of individual water-soluble vitamins. All methods were evaluated according to ICH guidelines and used for stability studies of water-soluble vitamins in the solid state, individually and in mixtures. Stability studies were performed in climatic chambers under various stress conditions - elevated temperature, relative humidity, light. We optimized sample preparation and adjusted the experimental conditions. Based on the results of a three-month stability study of individual vitamins at 60 °C and 75 % relative humidity, we confirmed adequate stability (> 90% of initial content) for most vitamins and lower stability (45 – 89 %) for vitamins B5, B12 and both forms of B2. In a one-week study of the exposure of individual water-soluble vitamins to light, we found out that all vitamins except vitamins C and B12 maintain their stability (> 90% of the initial content). Evaluation of the stability of water-soluble vitamin mixtures was difficult due to the inhomogeneity of the samples. However, we found out that the vitamins in the mixture are less stable than individually, which was also confirmed by organoleptic changes of samples (burned and resinous). The stability of the vitamins in the mixture was significantly affected by the higher relative humidity and its synergistic destabilizing effect with elevated temperature. We have successfully developed an analytical approach to evaluate the stability of individual water-soluble vitamins in the solid state. Further optimization of pre-analytical activities will be required to properly evaluate the stability of vitamins in mixtures.