Razvoj analizne metode za določanje fitoestrogenih spojin v pivu in hmelju

Kordež, May (author), Prosen, Helena (mentor)

Fitoestrogeni so sekundarni metaboliti rastlin, ki se zaradi strukturne podobnosti z ženskimi spolnimi hormoni estrogeni lahko vežejo na estrogenske receptorje in povzročijo odziv v telesu. Precej let so deležni velikega zanimanja s strani medicine in farmacije zlasti zaradi njihovih pozitivnih učinkov na človeško zdravje. Pojavljajo se v hmelju, ki je v povezavi s pivom osrednja tema tega dela. Namen magistrske naloge je bil razvoj postopka priprave vzorca za analizo fitoestrogenov v pivu in hmelju. Pivo sem razplinil na ultrazvočni kopeli in filtriral skozi filter papir, nato je sledila ekstrakcija na trdno fazo (SPE). Hmelj sem ekstrahiral z metanolom in ga filtriral skozi filter papir. Za optimizacijo ekstrakcije na trdno fazo sem poskuse izvajal z mešanico etanola in vode – »sintetično pivo«. Določil sem primerne pogoje: SPE kolona LC-8, 5 % dodatek NaCl, kisel pH vzorca (3-3,5) in največji volumen nanosa vzorca, ki je znašal 70 mL. Izkoristki ekstrakcije pri optimizirani SPE metodi so bili med 79 % in 88 %. Sledila je analiza s HPLC-DAD. Pri HPLC sem optimiziral pogoje ločbe, gradient in mobilno fazo, ki jo sestavljata ACN in voda, nakisana z 0,1 % mravljično kislino. Analite sem detektiral pri dveh različnih valovnih dolžinah: 290 nm, kjer imata absorpcijski maksimum 8-prenilnaringenin in izoksantohumol, ter pri 365 nm, kjer ima absorpcijski maksimum ksantohumol. Linearno območje je bilo med 0,05 mg/L in 50 mg/L, R2 so bili 0,9977 za ksantohumol, 0,9993 za isoksantohumol in 0,9994 za 8-prenilnaringenin. Rezultati analiz so bili ponovljivi z RSD med 0,98 % in 3,24 %. V vseh realnih vzorcih piva in hmelja sem določil ksantohumol in izoksantohumol, v nekaterih tudi 8-prenilnaringenin. Slednji se v večini vzorcev pojavlja v nizkih koncentracijah pod mejo zaznave HPLC-DAD metode. S tem namenom sem uporabil LC-MS/MS za potrditev prisotnosti 8-prenilnaringenina. Vsebovali so ga vsi obravnavani vzorci.

• Language: Slovenian
• Keywords: fitoestrogeni, pivo, hmelj, ksantohumol, izoksantohumol, 8-prenilnaringenin, HPLC-DAD, UHPLC-MS/MS
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: FKKT - Faculty of Chemistry and Chemical Technology
• Year: 2022
• PID: 20.500.12556/RUL-142268
• COBISS.SI-ID: 135121667
• Publication date in RUL: 28.10.2022

Secondary language

• Language: English
• Title: Development of analytical method for determination of phytoestrogenic compounds in beer and hops

Abstract

Phytoestrogens are secondary metabolites in plants. Due to their structural similarity to female sex hormones, estrogens, they have the ability to bind to estrogen receptors and can cause a response in the human body. For many years, phytoestrogens have been interesting from a medical as well as from pharmaceutical point of view especially because of their positive effects on human health. Phytoestrogens can be found in hops, and with its connection to beer, these two are the centre of this master’s thesis. The purpose of this thesis was to develop a procedure for sample preparation and the analysis of phytoestrogens in beer and hops. Beer samples were firstly degassed in an ultrasonic bath and then filtered through filter paper. Filtration was followed by solid phase extraction (SPE). Hop samples were firstly extracted with methanol and then filtered through filter paper. For solid phase extraction optimization, the experiments were performed with ethanol and water mixture, so called synthetic beer. I have set the appropriate extraction conditions: SPE column LC-8, 5 % NaCl, low sample pH (3-3,5) and the largest sample volume applied, 70 mL. Solid phase extraction yields were between 79 % and 88 %. After solid phase extraction a HPLC-DAD analysis took place. I have optimized separation parameters; gradient and composition of the mobile phase (acetonitrile and water with 0.1 % formic acid). Analytes were detected at two different wavelengths: 290 nm (absorption maximums of 8-prenylnaringenin and isoxanthohumol) and 365 nm (absorption maximum of xanthohumol). Concentration linearity range was 0.05–50 mg/L, R2 values ranged from 0.9977 to 0.9994 and the RSDs for repeatability ranged from 0.98 % to 3.24 %. In all the beer and hop samples xanthohumol and isoxanthohumol were determined, in some samples also 8-prenylnaringenin. The last one is present in most samples at low concentrations below the limit of detection of HPLC-DAD method. Therefore I used LC-MS/MS analysis and confirmed 8-prenylnaringenin in all of the samples.

• Keywords: phytoestrogens, beer, hops, xanthohumol, isoxanthohumol, 8-prenylnaringenin, HPLC-DAD, UHPLC-MS/MS