F. Cuyckens et al., Eur. J. Mass Spectrom. 9, 409–420 (2003) 409 The application of liquid chromatography-electrospray ionization mass spectrometry and collision-induced dissociation in the structural characterization of acylated flavonol O-glycosides from the seeds of Carrichtera annua Filip Cuyckens, a Abdelaaty A. Shahat, a Hilde Van den Heuvel, a Khaled A. Abdel-Shafeek, b Moustafa M. El-Messiry, b Medhat M. Seif-El Nasr, b Luc Pieters, a Arnold J. Vlietinck a and Magda Claeys a* a University of Antwerp (UA), Department of Pharmaceutical Sciences, Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail: claeys@uia.ua.ac.be b Department of Pharmaceutical Sciences, National Research Centre, 12311 Dokki, Cairo, Egypt The flavonoid fraction from the seeds of Carrichtera annua was studied using high-performance liquid chromatography simulta- neously coupled to a photodiode array detector (LC/UV-DAD) and a mass spectrometer equipped with an electrospray source (LC/ESI-MS). Collision-induced dissociation (CID) mass spectral data obtained off-line by nanospray (nano-ESI) analysis provided a wealth of complementary structural information, which was consistent with structures established by NMR or led to the proposal of base structures of the flavonol O-glycosides present in the Carrichtera annua seed extract. The flavonoid fraction was found to contain 12 structurally related flavonol O-glycosides. Eleven flavonoids, of which several were new compounds, were acylated with one or more benzoyl, feruloyl or sinapoyl groups. These acyl groups gave rise to characteristic product ions in the [M + H] + and [M + Na] + CID spectra as well as to radicalar acid-related product ions at high-energy collisional activation. In addition to the characterization of the acyl substituents, the mass spectral data allowed the identification of the aglycone, the determination of the base structure and the differentiation of several positional isomers. Keywords: Carrichtera annua, Cruciferae (Brassicaceae), acylated flavonol glycosides, liquid chromatography, mass spectrometry, electrospray ionization, collision-induced dissociation Introduction Flavonoid glycosides are predominant forms of natu- rally occurring flavonoids in plants, representing a large group of secondary plant metabolites. 1–3 They all contain a C 15 flavonoid as an aglycone and are usually divided into O- and C-glycosyl flavonoids. They are of interest because they have biological activities 4 and are useful chemotaxonomic marker compounds. 5,6 Recently, they have also received con- siderable interest as components of foodstuffs and nutraceuticals because of their antioxidant and anticancer properties. 7–9 Liquid chromatography coupled to electrospray ioniza- tion mass spectrometry (LC/ESI-MS) represents a very pow- erful tool for the analysis of natural products, since the mass spectrometer is a universal detector, which can achieve very high sensitivity and provide information on the molecular weight as well as on structural features. The first-order posi- tive and negative ESI mass spectra obtained by LC/MS on flavonoid O-glycosides, reveal a protonated and deproton- ated molecule, respectively, allowing a reliable determina- tion of the molecular weight, and also give mass information on the aglycone part. More detailed structural information can subsequently be obtained by resorting to collision- induced dissociation (CID) and tandem mass spectrometry (MS/MS). In the present work, low- and high-energy CID spectra were obtained using [M + H] + , [M + Na] + and [M – H] – ions as precursors by off-line nano-electrospray mass spectrometric analysis (nano-ESI-MS) on fractions isolated by analytical LC. The nano-ESI technique has the advantage that extensive and complementary mass spectral information can be obtained on very small amounts of iso- DOI: 10.1255/ejms.559 ISSN 1356-1049 © IM Publications 2003