Razvoj in validacija metode tekočinske kromatografije za kvantifikacijo kurkuminoidov v različnih topilih

Ribo, Aldina (author), Tavčar, Eva (mentor)

Kurkuminoidi so naravni lipofilni polifenoli, ki se pridobivajo iz korenike kurkume (Curcuma longa). V skupino kurkuminoidov spadajo demetoksikurkumin (DMC), bisdemetoksikurkumin (BDMC) in kurkumin (CUR). Slednji je najbolj raziskan in se veliko uporablja v prehranskih dopolnilih, kozmetiki ter kulinariki. Zaradi svojih antioksidativnih, protivnetnih, protibakterijskih in protirakavih lastnosti uporaba kurkumina v terapevtske namene vedno bolj narašča. Njegove koristne učinke omejujeta slaba topnost in slaba biološka uporabnost, zato poskušajo raziskovalci razviti načine za izboljšanje teh lastnosti. Za raziskovanje topnosti potrebujemo metodo, ki bo primerna za analizo kurkuminoidov v različnih farmacevtskih topilih, ki se uporabljajo v farmacevtskih oblikah kot pomožne snovi, kot tudi v različnih laboratorijskih topilih, ki se lahko uporabljajo za ekstrakcije in izolacije kurkuminoidov iz rastlinskih drog in farmacevtskih izdelkov. Naš namen v tej nalogi je bil razvoj in validacija metode HPLC za ugotavljanje vsebnosti kurkuminoidov v različnih vzorcih in analizo topnosti kurkumina (pri 22 °C) v diklorometanu, DMSO, etanolu, glicerolu, olju MCT in PEG 400 s stresanjem (»shake-flask« metoda). Razvili smo metodo, s katero lahko kvantificiramo posamezne kurkuminoide v različnih farmacevtskih topilih. Metodo smo razvili na reverznofazni koloni Phenomenex Kinetex C18 z izokratsko elucijo in kurkuminoide določali pri valovni dolžini 424 nm. Metoda traja 18 minut z gradientom mobilne faze B. Kurkuminoidi so se eluirali pri retencijskih časih: 6,31 min (BDMC), 7,24 min (DMC) in 8,30 min (CUR). Metodo smo validirali po smernicah ICH. Preverili smo linearnost metode, točnost, ponovljivost, selektivnost in mejo določljivosti. Pri ugotavljanju ustreznosti izdelkov iz kurkume na trgu smo ugotovili, da vsi izdelki ne ustrezajo specifikacijam njihovih proizvajalcev. Pri ugotavljanju primernosti metode za določanje topnosti kurkumina smo ugotovili, da je kurkumin najbolj topen v DMSO, sledi PEG 400, olje MCT, diklorometan in etanol. V nalogi smo dokazali, da HPLC metoda daje zanesljive in točne rezultate pri kvantifikaciji kurkuminoidov in da je primerna za ugotavljanje topnosti v različnih topilih.

• Language: Slovenian
• Keywords: kurkumin, kurkuminoidi, HPLC, validacija
• Work type: Master's thesis/paper
• Organization: FFA - Faculty of Pharmacy
• Year: 2023
• PID: 20.500.12556/RUL-152218
• Publication date in RUL: 11.11.2023

Secondary language

• Language: English
• Title: Development and validation of a liquid chromatography method for quantifying curcuminoids in various solvents

Abstract

Curcuminoids are natural lipophilic polyphenols obtained from the root of turmeric (Curcuma longa). The group of curcuminoids includes demethoxycurcumin (DMC), bisdemethoxycurcumin (BDMC) and curcumin (CUR). The latter is the most researched and widely used in food supplements, cosmetics and cuisine. Due to its antioxidant, anti-inflammatory, antibacterial and anti-cancer properties, the use of curcumin for therapeutic purposes is increasing. Its beneficial effects are limited by poor solubility and poor bioavailability, which are the subject to research for many scientists. To investigate solubility, we need a method that will be suitable for the analysis of curcuminoids in various pharmaceutical solvents used in pharmaceutical forms as excipients, as well as in various pharmaceutical solvents that can be used for the extraction and isolation of curcuminoids from herbal medicines and products. Our aim in this task was the development and validation of an HPLC method for the determination of curcuminoid content in different samples and the analysis of the solubility of curcumin (at 22 °C) in dichloromethane, DMSO, ethanol, glycerol, MCT oil and PEG 400 by shaking (»shake-flask« method). We have developed a method that can be used to quantify individual curcuminoids in various pharmaceutical solvents. The method was developed on a Phenomenex Kinetex C18 reverse phase column with isocratic elution and curcuminoids were determined at a wavelength of 424 nm. The method takes 18 minutes with a gradient of mobile phase B. Curcuminoids were eluted at retention times: 6.31 min (BDMC), 7.24 min (DMC) and 8.30 min (CUR). The method was validated according to ICH guidelines. We checked the linearity of the method, accuracy, reproducibility, selectivity and limit of quantification. When determining the suitability of turmeric products on the market, we found that not all products meet the specifications of their manufacturers. When determining the suitability of the method for determining the solubility of curcumin, we found that curcumin is the best soluble in DMSO, followed by PEG 400, MCT oil, dichloromethane and ethanol. In the assignment, we proved that the HPLC method gives reliable and accurate results in the quantification of curcuminoids and that it is suitable for determining solubility in various solvents.

• Keywords: curcumin, curcuminoids, HPLC, validation