A fragmentation study of an isoflavone glycoside, genistein-7-O-glucoside, using electrospray quadrupole time-of-flight mass spectrometry at high mass resolution

https://doi.org/10.1016/j.ijms.2004.01.001Get rights and content

Abstract

A mass spectrometric method based on the combined use of electrospray ionization, collision-induced dissociation and tandem mass spectrometry at high mass resolution has been applied to an investigation of the structural characterization of genistein-7-O-β-d-glucoside (5,7,4′-trihydroxyisoflavone). The product ion mass spectrum of [M−H] ions shows neutral losses of the glycan residue (162 Da) and of the glycan residue + Hradical dot (163 Da) by rearrangement and scission, respectively, where the latter loss dominates at higher collision energies. The genistein moiety remained intact and only minor fragmentation of the glucose moiety was observed. The low-energy product ion mass spectrum of [M+H]+ ions shows extensive fragmentation of the glucose moiety, though at low ion signal intensity, loss of the glycan residue, and simple fragmentation of the genistein moiety that permits characterization of the substituents in the A and B rings. The use of elevated cone voltages permitted observation of product ion mass spectra of selected primary fragment ions. Product ion mass spectra examined at high mass resolution allowed unambiguous determination of the elemental composition of fragment ions. Fragmentation mechanisms and ion structures have been proposed.

Introduction

The study of the class of phytochemicals known as the flavonoids has been confined largely heretofore to their distribution in the plant kingdom, elucidation of their structures, and the pathways by which they are synthesized. Flavonoid is a collective noun given to several classes of structurally similar, plant secondary metabolites; the major classes are flavones, isoflavones, flavans, anthocyanins, proanthcyanidins, flavanones, chalcones, and aurones. The flavonoids were reviewed extensively in 1994 [1]. Plants synthesize flavonoids along with other secondary metabolites for protection against pathogens and herbivores. Flavonoids are ubiquitous in the environment; they are found primarily in petals, the foliage of trees and bushes, and are widely distributed in the edible parts of plants.

Flavonoids and isoflavonoids may be of ecotoxicological importance since they are present in the heartwood of tree species used for wood pulp [2], [3] and are to be found in a variety of fruits and vegetables. Plants of the Leguminosae family (e.g. soy, lupin) contain isoflavones that are important components of the diets of humans and animals. Flavonoids are of environmental significance because several flavonoid aglycones (flavonoid glycosides that have lost the sugar moiety) are known to be biologically active [4] while some isoflavones are also phytoestrogens [5], [6] that have radical scavenger [7], [8] and anticarcinogenic [9], [10] activities. While phytochemicals in the heartwood and sapwood of trees render the wood resistant to disease [2], flavonoids in mammals can affect reproduction by acting upon the pituitary–gonadal axis, either as competitors for steroid receptor sites [11] or by inhibiting aromatase [12]. Flavonoids are present also in herbal medicines [13] and preventative therapeutics [9]. The potential health effects of flavonoids demand that methods be developed for their determination and quantification in food products, plant extracts, and serum.

The advent of fast atom bombardment (FAB), atmospheric pressure chemical ionization (APCI), and electrospray ionization (ESI) combined with tandem mass spectrometry (MS/MS) has permitted ready study of the flavonoids, their ion chemistry, and the determination of flavonoids in low concentrations in aqueous systems. Mass spectrometric methods coupled to liquid chromatography show great promise for the analysis and quantification of these compounds in biological samples [14], [15], [16], [17], food products [18], plant extracts [19], [20], [21], and for fundamental studies [22], [23], [24], [25], [26], [27], [28], [29]. Thermospray LC/MS/MS has been used for the characterization of flavonoids [30] and the rapid screening of fermentation broths for flavones [31]. Using ion spray LC/MS/MS, parent ion scans of the two protonated aglycones, quercitin and kaempferol, indicated the presence of more than 12 different flavonol glycosides among nine hop varieties [32]. Ion spray LC/MS/MS has been used also for the characterization of flavonoids in extracts from Passiflora incarnata [33], [34]. APCI/MS/MS has been employed for the quantitative analysis of xanthohumol and related prenylflavonoids in hops and beer [35], for a study of flavonone absorption following naringin, hesperidin, and citrus administration [36] and for the identification of 26 aglycones from the leaf surfaces of Chrysothamnus [37]. ESI/MS/MS has been employed for the investigation of 14 flavonoids [38], apigenin anionic clusters in the gas phase [39], Na+-bound clusters of quercetin in the gas phase [40], flavonoid aglycones [41], and characterization of flavonoid O-diglycosides [42], [43]. The combination of MS/MS with these ionization techniques for polar compounds has proven to be a valuable technique that has great sensitivity, specificity, mass range, and mass resolution.

Genistein-7-O-β-d-glucoside (5,7,4′-trihydroxyisoflavone) was selected for this study because of its wide range of biological activities both in plant physiology and biochemistry [7], [44], its estrogenic effect on women [5], [6], and anticarcinogenic properties [10]. Genistein is present in wood pulp and in treated and untreated pulp mill effluents, and it is probable that genistein and possibly other flavonoids are present in the receiving waters of pulp mills [38]. The investigation of genistein-7-O-glucoside forms a necessary base-line study for our current examination of flavonoids and flavonoid glycosides [38], [39], [40], [45]. This fragmentation study of genistein-7-O-glucoside consists of an examination of the fragmentation of protonated genistein-7-O-glucoside and of deprotonated genistein-7-O-glucoside formed by ESI. The mass/charge ratios of the fragment ions were obtained at high mass resolution to within 0.1 mDa. Pathways for the formation of primary fragment ions are proposed. For the observation of product ion mass spectra of primary fragment ions, signal ion intensities of primary fragment ions were maximized by variation of the cone voltage. Fragment ions generated at elevated cone voltages upstream of the first mass-resolving element can be subjected to CID so as to identify direct product ion–precursor ion relationships. The utilization of enhanced cone voltage to maximize first generation fragment ion signal intensity can yield pseudo-MS/MS/MS performance in the Q-TOF II™ mass spectrometer that is useful in determining the hierarchy of fragment ions. In the examination of a standard substance such as genistein-7-O-glucoside, the probability of isolating more than one isobaric fragment ion species in the first quadrupole mass filter is zero when such ions are not detected in the initial product ion mass spectrum.

Each of the primary ion species obtained thus was isolated in the mass-resolving quadrupole mass filter and fragmented further in the collision cell. This process of examination of primary, secondary, and tertiary ions was continued until prevented by lack of ion signal intensity. While the measurement of precise mass/charge ratios of fragment ions of low mass/charge ratio were difficult due to low signal ion intensity, each fragment ion reported was observed as a direct product of its immediate precursor ion. Ion structures have been proposed for each of the fragments observed in the fragmentation of the (Yo−H)radical dot ion of m/z 268.

Section snippets

Materials

Genistein-7-O-glucoside was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Methanol was purchased from Fisher (Fair Lawn, NJ, USA). The analyte solution was prepared using methanol and water (1:1) at a concentration of 100 μg ml−1. The solution was infused to the ESI source using a Harvard Apparatus Model 11 syringe pump (Harvard Apparatus, Holliston, MA, USA) at a flow rate of 10 μl min−1.

ES mass spectrometry

ES mass spectrometry and MS/MS experiments were performed on a Q-TOF 2™ mass spectrometer with a

Nomenclature

The nomenclature system for flavonoid aglycones, as developed by Mabry and Markham [46] for the definition of the various A and B ring fragments generated by EI, was found to be inadequate for describing the range of product ions formed when protonated molecules are subjected to CID [47], [48]. A more systematic ion nomenclature for flavonoid aglycones has been proposed [49] that is conceptually similar to that introduced for the description of carbohydrate fragmentations in product ion mass

Conclusion

Low-energy product ion mass spectra of the [M+H]+ and [M−H] ions of genistein-7-O-β-d-glucoside (5,7,4′-trihydroxyisoflavone) show simple fragmentation patterns that permit characterization of the substituents in the A and B rings. [M−H] ions show neutral losses of 162 and 163 Da by rearrangement and scission, respectively, leading to aglycone and radical aglycone product ion formation; radical aglycone formation dominates at higher collision energies. [M+H]+ ions also show a neutral loss of

Acknowledgements

The authors acknowledge the financial support from each of the Natural Sciences and Engineering Research Council of Canada (Discovery Grants Program), the Canada Foundation for Innovation, the Ontario Research & Development Challenge Fund and Trent University. The authors acknowledge with grateful thanks the guidance and counsel of a reviewer.

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