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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. Vlietincka and Magda Claeysa* University of Antwerp (UA), Department of Pharmaceutical Sciences, Universiteitsplein 1, B-2610 Antwerp, Belgium. E-mail:
[email protected] b Department of Pharmaceutical Sciences, National Research Centre, 12311 Dokki, Cairo, Egypt
a
The flavonoid fraction from the seeds of Carrichtera annua was studied using high-performance liquid chromatography simultaneously 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 naturally occurring flavonoids in plants, representing a large 1–3 group of secondary plant metabolites. They all contain a C15 flavonoid as an aglycone and are usually divided into Oand C-glycosyl flavonoids. They are of interest because they have biological activities4 and are useful chemotaxonomic marker compounds.5,6 Recently, they have also received considerable interest as components of foodstuffs and nutraceuticals because of their antioxidant and anticancer properties.7–9 Liquid chromatography coupled to electrospray ionization mass spectrometry (LC/ESI-MS) represents a very powerful tool for the analysis of natural products, since the mass spectrometer is a universal detector, which can achieve very
DOI: 10.1255/ejms.559
high sensitivity and provide information on the molecular weight as well as on structural features. The first-order positive and negative ESI mass spectra obtained by LC/MS on flavonoid O-glycosides, reveal a protonated and deprotonated molecule, respectively, allowing a reliable determination of the molecular weight, and also give mass information on the aglycone part. More detailed structural information can subsequently be obtained by resorting to collisioninduced 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-
ISSN 1356-1049
© IM Publications 2003
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lated product ( g amounts) without changing the sample. The small droplet size and the low nanoliter per minute flow rate provide a very long time for tandem mass spectrometric experiments with only limited product consumption10. Concerning complementary mass spectral information, different precursor ions can be selected, the positive or negative ion mode can be chosen and the collision-induced dissociation can be performed at both low- and high-energy. Optimized LC/MS methods based on electrospray ionization (ESI) in the positive and negative ion modes have recently been reported for the analysis of flavonoids.11–13 In the method developed in our own laboratory,13 a reversed phase LC column is also coupled to a UV-photodiode array detector (DAD); in this way, UV spectra are obtained, which are structurally informative since flavonoids show charac3 teristic UV absorbances. Although NMR techniques are preferred for structure elucidation of flavonoid glycosides, mass spectrometric techniques can make a valuable contribution since they can be applied to much smaller quantities of isolated compounds. Especially in the case where major compounds have already been characterized by NMR, mass spectrometry can significantly speed up the identification of related compounds. With regard to structure characterization of flavonoid O-glycosides, structural information can be obtained on: (1) the aglycone part,14–16 (2) the types of carbohydrates present (mono-, di-, tri- or tetrasaccharides and hexoses, deoxyhexoses or pentoses),17 (3) the stereochemical assignment of terminal monosaccharide units,18 (4) the sequence of the glycan part,19 (5) interglycosidic linkages20,21 and (6) attachment points of carbohydrate residues to the aglycone.21–26 In addition, we will demonstrate how mass spectrometry can provide structural information on the identity of acyl substituents. In the present study, we have applied LC/MS and CID MS/MS techniques to the characterization of the flavonoid fraction of the seeds of Carrichtera annua. Carrichtera annua (L.) DC is an annual herb growing up to 40 cm, which is covered with bristly hairs. It belongs to the Cruciferae (or Brassicaceae) and is a native plant of the Mediterranean region. Plants of the Cruciferae are used in traditional medicine for the treatment of many diseases, such as cancer, rheu27–29 matism, diabetes and bacterial and fungal infections and Carrichtera annua in particular is known to be used by the native Bedouins as an antidiabetic and antispasmodic. Flavonoids are a major group of constituents and are assumed to be among the beneficial components. In a previous study, a new acylated flavonol triglycoside, i.e quercetin 3-O-[(6-feruloyl- -glucopyranosyl)-(1 2)- -arabinopyranoside]-7-O- -glucopyranoside, was reported from the upper part of Carrichtera annua.30 The flavonoid fraction of the seeds of Carrichtera annua was found to contain 12 flavonol O-glycosides, of which 11 were acylated with one or more benzoyl, feruloyl and/or sinapoyl groups. The 12 flavonoids were characterized as completely as possible with LC/MS and CID MS/MS
Structural Characterization of Acylated Flavonol O-Glycosides
techniques, while one compound was fully characterized with the help of NMR.
Experimental Plant material and extraction of flavonoids for MS analysis
Carrichtera annua was collected from El-Araish, North Sinai Egypt in March 1998 and identified by Professor Dr N. Elhadidi, Department of Plant Taxonomy and Flora, Faculty of Sciences, University of Cairo, Cairo, Egypt. A voucher specimen (collection no. 796) has been deposited at the herbarium of the National Research Centre, Cairo, Egypt. 10 mg of an 80% aqueous methanol extract of the ground powdered seeds of Carrichtera annua was purified on a preconditioned C18 cartridge (200 mg adsorbent, 3 mL, Bond Elut®, Varian, Harbor City, CA, USA). After washing the cartridge three times with 4 mL of water, the flavonoids were eluted with 50% methanol and dried. This purified extract was used for LC/MS, LC/UV and MS/MS analysis. Extraction and isolation of compound 7 for NMR analysis
Thin layer chromatography was carried out on precoated Silica gel F254 plates (Merck, Darmstadt, Germany) developed with ethyl acetate–acetic acid–formic acid–water (100 : 10 : 10 : 20). Spots were detected using Neu’s spray reagent (1% diphenylboric acid ethanolamine complex in methanol).3 Column chromatography was performed on polyamide 6 S Riedel-de Haën, Silica gel (Merck) and Sephadex LH-20 (Pharmacia) (Merck) (Darmstadt, Germany). The ground powdered seeds of Carrichtera annua (100 mg) were defatted with petroleum ether (40–60°C), and extracted with aqueous methanol (80%). The extract was concentrated to remove the methanol and transferred to a polyamide column (polyamide was pre-swollen in distilled water). The column was washed with 250 mL water and the flavonol glycosides were eluted with 250 mL methanol. After concentration the residue was chromatographed with methanol on a Sephadex LH-20 column. The first fraction (50 mL) was discarded and the second fraction (70 mL) showed five spots on TLC. Compound 7 was obtained by repeated column chromatography on Sephadex LH-20 with methanol as eluent. LC/UV and LC/MS analysis
A Waters 600 MS pump (Waters, Milford, MA, USA) was simultaneously connected to a Waters 991 photodiode array detector and an Autospec-oa-Tof mass spectrometer (Micromass, Manchester, UK), equipped with an electrospray ionization (ESI) source, by a laboratory-made splitter with a split ratio of 1 : 50. The chromatographic separation was carried out on a Waters Xterra RP-18 MS column (5 m, 250 × 3 mm), preceded by a Waters Xterra C18 MS guard column (20 × 3 mm). 10 L of the Carrichtera annua seed extract was dissolved in methanol/water (1 : 1; v/v) and
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injected. The mobile phase consisted of (A) water and (B) acetonitrile, both containing 0.5% (v/v) formic acid. A linear gradient of 12–25% B over 25 min was used, after which the percentage B was increased to 100% over 6 min. All solvents were filtered through a 0.22 m Nylon 66 membrane (Alltech, Deerfield, IL, USA) and were continuously degassed by a 50 mL min–1 helium flow. On-line UV spectra were recorded between 220 and 400 nm (1 scan s–1) with sampling points every 5.2 nm using the photodiode array detector. Mass spectrometric data were obtained on an Autospec-oa-ToF mass spectrometer with the ESI source operated at 4 kV and at a cone voltage of 40 V. Nitrogen was used both as bath gas (80°C) and nebulizing gas.
drate residue, product ions are designated as Bi, where i represents the number (ε1) of the glycosidic bond cleaved counting from the non-reducing terminus. The nomenclature proposed by Ma et al.14 has been followed to denote the product ions resulting from aglycone fragmentation. m,nA and 0 m,n B0 labels are used to designate product ions containing intact A and B rings, respectively, in which the superscripts m and n indicate the C-ring bonds that have been broken. The subscript 0 to the right of the letter is used to avoid confusion with the Ai and Bi (i ε 1) labels that are used to designate carbohydrate fragments. To avoid confusion with a protonated aglycone, Y0 (Na) is used to denote a sodium-containing aglycone ion.
Nano-ESI-MS
NMR analysis 1
Nano-ESI-MS analysis was performed on fractions containing individual peaks that were collected during three analytical LC runs (about 2–50 g of each compound). Mass spectra were obtained on the Autospec-oa-Tof instrument with a nano-ESI probe by direct infusion with a syringe pump (Harvard apparatus syringe infusion pump 22; Harvard, South Natick, MA) employing a 100 L syringe (Hamilton, Reno, NV) at a constant flow rate of 150 nL min–1.The compounds were dissolved in water/methanol (30 : 70; v/v) at a concentration of about 10 M. In the MS/MS mode precursor ions were selected by MS1 (EBE configuration) and after passing the collision cell, product ions were recorded on the microchannel plate detector of the time-of-flight analyzer. Low- and high-energy CID spectra were obtained using helium and xenon, respectively, as collision gas until 75% attenuation of the precursor ion beam. Data acquisition and processing were performed using OPUS V3.1X software. All scans were acquired in the continuum mode. The nomenclature proposed by Domon and Costello31 for glycoconjugates has been adopted to denote the product j and Yj, k,l ions. Ions containing the aglycone are labeled X where j is the number of the interglycosidic bond broken, counting from the aglycone and the superscript k and l indicate the cleavages within the carbohydrate rings. The glycosidic bond linking the glycan part to the aglycone is numbered 0. When the charge is retained on the carbohy-
13
H and C NMR as well as two-dimensional (COSY, HSQC, HMBC) NMR spectra were recorded in MeOH-d4 on a Bruker DRX-400 spectrometer operating at 1 13 400.13 MHz for H and at 100.61 MHz for C. Chemical shifts are presented in ppm downfield of tetramethylsilane.
Results and discussion Figure 1 presents the (a) LC-UV and (b) LC-ESI/MS chromatogram obtained in the negative ion mode for the Carrichtera annua seed extract and (c) the UV spectrum recorded for compound 2. The UV spectrum shows two absorption maxima: one around 256 nm (band II) and another around 331 nm (band I), which are characteristic for flavones and flavonols.3 A dip in the UV spectrum can be noted around 280 nm that is typical for a flavonol structure, while the lower band I absorption (
