Razvoj analizne metode na osnovi natrijevega borohidrida in tekočinske kromatografije visoke ločljivosti za vrednotenje koencima Q10

Štagar, Nina (author), Roškar, Robert (mentor)

Na slovenskem tržišču je prisotnih veliko prehranskih dopolnil in eno zdravilo, ki vsebujejo oksidirano ali reducirano obliko koencima Q10. Pogosto pa ti izdelki vsebujejo tudi druge aktivne sestavine, kot vitamin E in E-acetat, ?-karoten in askorbinsko kislino. Povpraševanje po teh izdelkih narašča, saj koencim Q10 izkazuje številne pozitivne učinke. Zato je zelo pomembno, da imamo na razpolago ustrezne analizne metode za preverjanje kakovosti tovrstnih izdelkov. Namen magistrske naloge je bil vpeljati nov način za določanje skupne koncentracije koencima Q10, in sicer z redukcijo z natrijevim borohidridom. Optimizirali smo ključne stopnje redukcije, pomemben del pa je predstavljala tudi izbira končnega topila z namenom kratkoročne stabilizacije reducirane oblike koencima Q10. Razviti postopek smo primerjali z uveljavljenim postopkom redukcije z askorbinsko kislino. Ugotovili smo, da je uspešnost redukcije po obeh postopkih primerljiva, a je metoda z askorbinsko kislino dolgotrajna in ni primerna za redukcijo koencima Q10 v izdelkih. Preizkusili smo tudi različna topila in antioksidante za stabilizacijo reducirane oblike Q10 in ugotovili, da je najprimernejše topilo za zagotavljanje kratkoročne stabilnosti (stabilnost vsaj dva dni) askorbinska kislina v acetonitrilu (molsko razmerje askorbinska kislina proti koencim Q10 je 1 proti 5) po redukciji z natrijevim borohidridom. Na zdravilu s koencimom Q10 – Fidi koencim 10 smo nato preverili razviti postopek določitve skupne vsebnosti koencima Q10 na osnovi redukcije. V ta namen smo preverili in optimizirali predhodno razvito metodo za ekstrakcijo koencima Q10 in drugih lipofilnih vitaminov iz mehkih kapsul. Za vrednotenje vsebnosti pa smo uporabili metodo na podlagi tekočinske kromatografije visoke ločljivosti (HPLC), sklopljene z UV detektorjem, ki smo jo prilagodili za naš namen ter jo ovrednotili za obe obliki Q10 v skladu s smernicami za validacijo analiznih metod. Dodatno pa smo v primerjavo določitve skupnega koencima Q10 vključili še dva načina, in sicer pretvorbo v popolnoma oksidirano obliko koencim Q10 ter sočasno določitev posameznih oblik (oksidirane in reducirane) koencima Q10. Ugotovili smo, da so vse tri metode primerljive in primerne za določitev skupne vsebnosti koencima Q10. Vsebnost določenega skupnega koencima Q10 v testiranem izdelku se je zelo dobro ujemala z navedeno vrednostjo.

• Language: Slovenian
• Keywords: koencim Q10, HPLC, redukcija, natrijev borohidrid
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-109641
• Publication date in RUL: 06.09.2019

Secondary language

• Language: English
• Title: Development of an analytical method for determination of coenzyme Q10 by sodium borohydride and high performance liquid chromatography

Abstract

On the Slovenian market, there are many dietary supplements and one registered medicine containing an oxidized or reduced form of coenzyme Q10. Often, other active ingredients are included, among others vitamin E and E-acetate, ?-carotene and ascorbic acid. Demand for these products is rising, because coenzyme Q10 shows a number of positive effects. Therefore, it is very important that we have appropriate analytical methods for quality control of such products. The purpose of this master’s thesis was to introduce new way of determining the total concentration of coenzyme Q10 by reduction with sodium borohydride. We optimized key steps of reduction and important part was also choosing final solvent with the aim of short-term stabilization of the reduced form of coenzyme Q10. Developed procedure was then compared with the established procedure of reduction with ascorbic acid. We found that the reduction is comparable, with difference that the ascorbic acid method is long-lasting and it’s not suitable for the reduction of coenzyme Q10 in products. We also tested various solvents and antioxidants for stabilization of reduced form of coenzyme Q10 and found that the best solvent for the short-term stabilization (stabilization at least 2 days) is ascorbic acid in acetonitrile (molar ratio ascorbic acid against coenzyme Q10 is 1 to 5) after reduction with sodium borohydride. Then we tested developed procedure of determination of total content of coenzyme Q10, based on reduction, on medicine with coenzyme Q10 – Fidi koencim 10. Therefore, we also verified and optimized pre-developed method for the extraction of coenzyme Q10 and other lipophilic vitamins from soft capsules. For evaluation of content we used method, based on high performance liquid chromatography coupled with UV detector, that we adapted for our purpose and evaluated it for both forms of coenzyme Q10 according to guidelines of validation of analytical methods. Additionally, in comparison, we included 2 more ways to determine total coenzyme Q10, namely conversion to oxidized form of coenzyme Q10 and determination of individual forms (oxidized and reduced) at the same time. We have found that all three methods are comparable and suitable for determining the total content of coenzyme Q10. Determined content of total coenzyme Q10 in tested product very well matched labeled value.

• Keywords: coenzyme Q10, HPLC, reduction, sodium borohydride