Razvoj ekstrakcijskega postopka za določanje hidrofilnih in lipofilnih vitaminov iz multivitaminskih izdelkov

Horvat, Andreja (author), Roškar, Robert (mentor), Temova Rakuša, Žane (comentor)

V multivitaminskih izdelkih se pogosto nahajajo hidrofilni in lipofilni vitamini, ki se zelo razlikujejo v fizikalno-kemijskih lastnostih. Ker v literaturi ne najdemo postopkov za enostavno in hkratno pripravo vzorcev za analizo obeh skupin vitaminov iz izdelkov, smo v ta namen razvili ekstrakcijske postopke za sočasno ločevanje hidrofilnih in lipofilnih vitaminov. Vrednotili smo deset hidrofilnih (askorbinska kislina, tiaminijev klorid, riboflavin, nikotinamid, kalcijev pantotenat, piridoksinijev klorid, biotin, folna kislina, cianokobalamin in tetrahidrofolna kislina) in pet lipofilnih (retinil acetat, retinil palmitat, D3, K1, tokoferil acetat) vitaminov, ki se najpogosteje pojavljajo v multivitaminskih izdelkih. Pred razvojem ekstrakcijskega postopka smo za namene vrednotenja vitaminov preverili ustreznost dveh predhodno razvitih kromatografskih metod, ene za lipofilne (metoda HPLC) in druge za hidrofilne vitamine (metoda UHPLC). Metodi smo ovrednotili v skladu s smernicami ICH in za obe potrdili selektivnost, linearnost, točnost, ponovljivost ter stabilnost analitov med analizo. Razvoj in optimizacijo ekstrakcijskega postopka smo najprej izvedli na vodnih zmeseh standardov, nato pa še na izbranih izdelkih različnih farmacevtskih oblik. Postavili smo tri ločene ekstrakcijske postopke, in sicer za sirupe, kapsule in tablete, pri čemer smo pri optimizaciji preverjali različne ekstrakcijske parametre: vrstni red uporabe topil, izbira vodne in organske faze, njun volumen in razmerje, število ciklov ekstrakcije, čas mešanja in stresanja, odvzem vzorca in druge. Z optimiziranimi postopki smo uspešno ekstrahirali obravnavane vitamine iz vseh treh farmacevtskih oblik, kar smo ovrednotili s ponovljivimi (RSD < 5%) in visokimi izkoristki ekstrakcije (100 ± 10%) za večino analitov. Ugotovljene vsebnosti vitaminov v izbranih izdelkih so bile v večini primerov višje od navedenih, a le izjemoma izven mej sprejemljivosti, ki veljajo po priporočilih USP za prehranska dopolnila. Razvite postopke za sočasno ekstrakcijo vitaminov smo nato primerjali s predhodno razvitimi ločenimi postopki za obe skupini vitaminov in dobili primerljive rezultate. Zaključimo lahko, da so vpeljani postopki priprave vzorca primerni za analizo vitaminov v multivitaminskih izdelkih, pri čemer je glavna prednost enkratna priprava vzorca za obe skupini vitaminov, kar skrajša čas izvedbe in zmanjša porabo vzorca in topil za analizo.

• Language: Slovenian
• Keywords: hidrofilni vitamini, lipofilni vitamini, HPLC, UHPLC, ekstrakcija, vsebnost
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2019
• PID: 20.500.12556/RUL-112943
• Publication date in RUL: 23.11.2019

Secondary language

• Language: English
• Title: Development of an extraction procedure of hydrophilic and lipophilic vitamins from multivitamin products

Abstract

Water-soluble and fat-soluble vitamins are very different in their physicochemical properties, but are often found together in multivitamin products. Our main goal was to develop simple procedures for the simultaneous extraction and separation of water- and fat-soluble vitamins, because such sample of preparation for the analysis of both vitamin groups cannot be found in the other literature. We evaluated ten hydrophilic vitamins (ascorbic acid, thiamine hydrochloride, riboflavin, niacinamide, calcium pantothenate, pyridoxine hydrochloride, biotin, folic acid, cyanocobalamin and tetrahydrofolic acid) and five lipophilic vitamins (retinyl acetate, retinyl palmitate, D3, K1, alpha tocopheryl acetate) in their forms, most commonly found in multivitamin products. Two previously developed chromatographic methods (HPLC method for fat-soluble and UHPLC method for water-soluble vitamins) were verified in accordance with ICH guidelines, before the development of extraction procedures, confirming their selectivity, linearity, accuracy, repeatability and sample stability for both vitamin groups. The development of the extraction procedures was first performed on aqueous standards mixtures and later also on selected commercial products in different dosage forms. For this purpose, three different extraction procedures were developed for each dosage form (capsules, tablets and oral solutions). During extraction procedure optimization, the following parameters were verified: solvent addition order, choice of aqueous and organic solvent, their volumes and ratios, number of extraction cycles, mixing and shaking time, sampling and others. The developed final extraction procedures were suitable for vitamins extraction from each tested dosage forms. It was then confirmed by high extraction efficiency (100 ± 10%) and low variability of the results (RSD < 5%) for most analytes. The determined vitamin contents in the tested products were typically higher than the labeled contents, but were seldom beyond the acceptable USP limits for dietary supplements. The developed extraction procedures were further compared with established extraction procedures of the separate vitamin groups. The obtained results were equivalent or even better. Therefore it can be concluded that the developed extraction procedures are suitable for the analysis of both vitamin groups in multivitamin products, with a single sample preparation as the main advantage, thus reducing the time and sample usage as well as solvents for analysis.

• Keywords: water-soluble vitamins, fat-soluble vitamins, HPLC, UHPLC, extraction, assay