Vitamins are a group of organic compounds with many specific functions in the human body. They are divided into two main groups: hydrophilic (water-soluble) vitamins - C and B complex vitamins and lipophilic (fat-soluble) vitamins - A, D, E, K. Typically vitamins cannot be synthesized in our body and are thus considered as essential micronutrients. Adequate intake and maintenance of optimal vitamin status in the body are essential for maintaining health at all life stages. Nowadays, due to the modern way of life and obtaining food, which usually yields lower vitamin amounts, vitamin deficiencies are becoming increasingly common. As a result, there is a growing use of vitamin supplements. These can be found in a very wide range of foods, food supplements, or medicines on the Slovenian market. Aiming to achieve the desired effects, their users rely on the quality of such products, which depends on their manufacturers, as well as the regulatory authorities% requirements and control of individual product categories. One of the biggest challenges in ensuring the proper quality of vitamin products is vitamins' instability, which is commonly known but insufficiently researched in detail. Within this doctoral thesis, we focused on the development of an appropriate analytical methodology for investigations of vitamins' stability, approaches for their stabilization, and quality control of a wide range of (multi)vitamin products on the Slovenian market. Due to very different physicochemical properties, we have developed separate analytical methods for the evaluation of lipophilic and hydrophilic vitamins. The analytical evaluation of lipophilic vitamins (A, D, E, K in [beta]-carotene) also covered coenzyme Q10 (CoQ10), which is often present in finished products with lipophilic vitamins. The hydrophilic vitamins' group included all their major forms (ascorbic acid, thiamine, riboflavin, nicotinamide, calcium pantothenate, pyridoxine, biotin, folic acid, and cyanocobalamin) and two additional vitamins (riboflavin sodium 5-phosphate and dexpanthenol). The presented methods represent a novelty in the field of vitamin analysis, as they provide a comprehensive and simultaneous evaluation of all lipophilic vitamins and CoQ10 or all hydrophilic vitamins, including the analytically most demanding vitamins. Their additional key advantages over other published analytical methods are simplicity, speed, cost-effectiveness, suitability for simultaneous evaluation of vitamins' stability, and applicability for routine use. Moreover, we took a step forward in the analytical evaluation of products containing individual vitamins, whose number on our market is constantly growing. Thus, we provided stability-indicative methods for vitamin D3, both forms of CoQ10 (ubiquinone and ubiquinol) and retinoids (retinol, retinyl acetate, retinyl palmitate, and [beta]-carotene). The presented methods were developed by selecting appropriate chromatographic conditions on high-performance liquid chromatography systems with different detection (DAD for the simultaneous evaluation of hydrophilic vitamins and UV for evaluation of all other vitamins). In addition, liquid chromatography coupled with mass spectrometry was used to identify transformation products formed during vitamins' degradation (e.g. vitamin D3). All presented methods were properly evaluated following the ICH guidelines for analytical methods' evaluation, thus confirming their suitability for further use. In addition to the instrumental analytical methods, we have also developed simple and fast extraction procedures for the evaluation of vitamins' contents in finished products. The comprehensive analytical methodology was optimized for the specific evaluation of vitamins in solutions and various finished products in the presence of many potential interferences (other vitamins, their degradation products, excipients, and impurities). Appling the developed methods for the analysis of many (multi)vitamin products, we confirmed their usefulness for routine analysis of finished products. The presented analytical methodology was further applied for the systematic evaluation of vitamins' stability in systems of increasing complexity, starting with solutions. Within the forced degradation studies, performed following the ICH Q1A and Q1B guidelines, we found that vitamins A, C, D, E, and 5-methyltetrahydrofolic acid (the active form of folic acid) are very sensitive to all tested stress conditions (elevated temperature, light, hydrolytic and oxidative conditions). Individual stress conditions strongly affected the stability of other vitamins. Vitamin B6 (pyridoxine) and B3 (nicotinamide) were identified as the most stable vitamins. We quantitatively evaluated the influence of various factors (e.g. medium, concentration, light, oxygen, temperature, pH, hydrogen peroxide, presence of other vitamins) on the stability of individual vitamins using chemical kinetics. Furthermore, we continued with the stability evaluation of selected individual vitamins in more complex systems, including commercially available medicines and food supplements (vitamin D3 and CoQ10), and cosmetics (retinoids). Vitamin D3 instability and degradation were also observed in the tested products, which provided different vitamin D3 stabilization. In general, the tested medicines provided better stability of vitamin D3 than the food supplements, in which significant, and even complete vitamin D3 degradation was observed during 6 months of storage at room temperature. The determination of the shelf-life after opening of multidose products brings an additional value to this study, as it is important information for users of such products but is rarely defined by their manufacturers. Evaluating the stability of CoQ10 in 11 products, we observed its degradation and also conversion between the two CoQ10 forms. Namely, the oxidized CoQ10 form was converted to the reduced form. However, the reduced CoQ10 form, which is recognized as the less stable form, was properly stabilized in the products. For further clarification of the observed CoQ10 reduction in the tested products, we additionally evaluated its stability in simpler systems - CoQ10 standard solutions with the addition of various antioxidants. Thereby, vitamin C was identified as a key factor for CoQ10 reduction in soft-shell capsules. Further on, we revealed significant retinoid instability in the 12 tested cosmetic products. In these, we detected up to 80% decrease in retinoid contents after six months of storage under ordinary conditions. The instability of retinoids in the tested products was even more pronounced under the influence of elevated temperature or light. Based on the data, obtained within the comprehensive and systematic evaluation of vitamins' stability in different systems, we can conclude that vitamin instability is an important matter, and proper vitamin stabilization is fundamental for the formulation of quality products with a longer shelf life. With the objective to ensure appropriate quality and stability of vitamins' products from their production to the end-user, we further investigated various approaches towards their stabilization. Thereby, we focused on vitamins, which were considerably unstable during the stability testing in solutions and products. We approached the stabilization of lipophilic vitamins by adding different concentrations of various stabilizers. Among these, EDTA was the most effective, as it stabilized vitamins D3, E and K by more than 20-fold. Thoroughly investigating approaches to stabilize aqueous vitamin D3 solutions, we found that these can be significantly stabilized (by at least 2-fold) by the selection of proper aqueous medium (Milli-Q water), storage at lower temperatures, and pH adjustments (above pH 5). The research work on vitamins' stabilization was further upgraded with technological approaches. We aimed to stabilize incompatible active agents; i.e., salicylic acid and vitamin D3, whose derivates are used in psoriasis treatment. In cooperation with the Chair of Pharmaceutical Technology, Faculty of Pharmacy, we incorporated salicylic acid and vitamin D3 into a properly formulated drug delivery system - a medicated foam. Thus we achieved significant vitamin D3 stabilization in the presence of salicylic acid (30-fold lower degradation constant compared to their solution). The formulation of a propellant-free foam, with sufficient foaming properties, and low irritancy potential offers an innovative drug delivery system for further translation into clinical practice of psoriasis treatment. The final part of the doctoral thesis was the quality evaluation of (multi)vitamin products on the Slovenian market. For such purpose, we quantitatively evaluated a total of 570 vitamins' contents in 155 (multi)vitamin products, including 11 medicines, 43 food supplements, 55 foods, and 46 cosmetic products. The obtained results reveal a high variability between the actual and labelled vitamins' contents in the individual products. We also found a correlation between the observed content deviations and the stability of individual vitamins. As expected, the deviations from the label claims were most pronounced in food supplements and food products. The determined vitamins' contents were higher than labelled in most of the tested products. These overages accounted mostly for up to 130%, but also up to 900% of the label claims, which poses a risk of excessive vitamin intake. The latter may be associated with serious health consequences, especially in the case of vitamins A and D. On the other hand, the observed absence of the labelled vitamins or their presence in a significantly lower amount than declared may be the reason for their insufficient intake to meet the needs. We believe that the results of our extensive research represent important information not only for the professionals but also for consumers and will contribute to greater awareness of the current situation of (multi)vitamin products on the Slovenian market. With the development of the comprehensive analytical methodology, we managed to overcome the current limitations of the published analytical methods and offer a tool for the time- and cost-effective evaluation of vitamins' stability. At the same time, considering the simplicity and accessibility of the equipment, the presented methods are also suitable for routine analysis of finished products. The identified irregularities in vitamins' contents and their instability in the tested products reveal the need for their proper stabilization as well as greater control and the establishment of stricter legislation in the field of food, food supplements, and cosmetics.
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