Naslov
Development of an LC-DAD-MS Method for Simultaneous Determination of Flavonoid Aglycones and their Metabolites

Autori
Bojić, Mirza ; Benković, Goran ; Antolić, Andrea ; Maleš, Željan

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, znanstveni

Izvornik
HPLC 2018 - 47th International Symposium and Exposition on High Performance Liquid Phase Separations and Related Techniques - Final Program / Dovichi, Norm - Washington (MD) : Barr Enterprises, 2018

Skup
47th International Symposium and Exposition on High Performance Liquid Phase Separations and Related Techniques (HPLC 2018)

Mjesto i datum
Washington D.C., Sjedinjene Američke Države, 29.07.2018. - 03.08.2018

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
flaovonoids, HPLC
(flavonoids, HPLC)

Sažetak
Flavonoids are ubiquitous compounds found in vegetables and fruits that have been extensively studied due to their diverse biological effects that could have positive impact on human health. Backbone of flavonoid structure is phenylbenzopyran that is hydroxylated and hydroxyl groups can further be methylated or bound to sugar moieties contributing to the diversity of this class of compounds. Data on metabolism of flavonoids is limited and mostly available to handful of flavonoids that have been extensively studied. Thus, the objective of this study was to develop LC-DAD-MS method for simultaneous determination of flavonoid aglycones and their metabolites. Metabolism of 30 selected flavonoids was determined using recombinant cytochrome P450 as well as human liver microsomes. After the incubations, reactions were stopped by addition of organic solvent (acetonitrile), mixed and centrifuged. Clear supernatant was used for analysis. Separation was achieved on Poroshell 120 EC-C18 column (100 x 3 mm, 2.7 µm) at 40 °C with a gradient method using water:methanol:formic acid in volume ratios of 93:5:2 and 3:95:2 as mobile phase A and B, respectively. Following gradient was used (t/min, %B): (0, 40), (14, 80), (15, 80), (16, 40), (20, 40) at a flow rate of 0.4 mL/min. High resolution mass spectrometry was used for identification and diode array detector for quantification of metabolites. Out of 30 flavonoids metabolism mediated by cytochromes P450 was observed for 15 of them, successfully identifying all metabolites. Several flavonoids showed competing and processive reactions of demethylation and hydroxylation, e.g. tangeretin, an O-pentamethoxylated flavone, has five positions susceptible to demethylation and can be hydroxylated at one additional position. Flavonoids are mainly susceptible to the hydroxylation (gain of oxygen, m/z 15.995) and demethylation (loss of methylene, m/z -14.016) reactions. While HRMS provided data on accurate mass it is important to note that developed HPLC method was able to separate and thus discriminate structural isomers based on retention time.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija, Farmacija

POVEZANOST RADA