Identification of the Position of Mono-O-glucuronide of Flavones and Flavonols by Analyzing Shift in Online UV Spectrum (λmax) Generated from an Online Diode Array Detector

Abstract

The beneficial pharmacological effects of flavonoids such as chemoprevention against cancer, aging, and heart diseases are severely limited due to their extensive in vivo glucuronidation by UDP-glucuronosyltransferases (UGTs). UGTs showed regiospecificity (i.e., position preference) in the glucuronidation of the flavonoids based on the substrate's chemical structure. In this paper, glucuronide(s) of 36 flavones and flavonols were generated using an in vitro glucuronidation reaction. UPLC/MS/MS was used to confirm the degree (mono- or di-) of glucuronidation in flavonoids with up to four hydroxyl groups. UV spectra of flavonoids and their respective mono-O-glucuronides were generated using UPLC with an online diode array detector. Analysis of the extent of shift in spectra of glucuronides in band I (300-385 nm) and band II (240-280 nm) regions as reflected by changes in lambdamax value was used to identify the position of glucuronidation. The data showed that glucuronidation of the 3- and 4'-hydroxyls resulted in band I lambdamax hypsochromic shifts (or blue shift) of 13-30 and 5-10 nm, respectively. Glucuronidation of the 5-hydroxyl group caused a band II lambdamax hypsochromic shift of 5-10 nm. In contrast, glucuronidation of the 7-hydroxyl group did not cause any lambdamax change in band I or II lambdamax, whereas glucuronidation of the 6-hydroxyl group did not cause predictable changes in lambdamax values. The paper demonstrated for the first time that a rapid and robust analysis method using lambdamax changes in online UV spectra can be used to pinpoint region-specific glucuronidation of flavones and flavonols with hydroxyl groups at the 4'-, 3-, 5-, and/or 7-position(s).