Differentiation of Flavonoid Glycoside Isomers by Using Metal Complexation and Electrospray Ionization Mass Spectrometry
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View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Differentiation of Flavonoid Glycoside Isomers by Using Metal Complexation and Electrospray Ionization Mass Spectrometry Michael Pikulski and Jennifer S. Brodbelt Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, Texas, USA The elucidation of flavonoid isomers is accomplished by electrospray ionization tandem mass spectrometry (ESI-MS/MS) via formation and collisional activated dissociation (CAD) of metal/flavonoid complexes containing an auxiliary ligand. Addition of a metal salt and a suitable neutral auxiliary ligand to flavonoids in solution results in the formation of [M(II) ϩ (flavonoid-H) ligand] complexes by ESI which, upon collisional activated dissociation, often result in more distinctive fragmentation patterns than observed for conventional protonated or deprotonated flavonoids. Previously, 2,2Ј-bipyridine was used as an auxiliary ligand, and now we compare and explore the use of alternative pyridyl ligands, including 4,7-diphenyl-1,10- phenanthroline. Using this technique, three groups of flavonoid glycoside isomers are differentiated, including glycosides of apigenin, quercetin, and luteolin. (J Am Soc Mass Spectrom 2003, 14, 1437–1453) © 2003 American Society for Mass Spectrometry lavonoids are polyphenolic phytochemicals ivatization was required to make the flavonoids suffi- which occur in edible fruits and vegetables. They ciently volatile for EI and in some cases did not result in Fhave been reported to act as antioxidants [1], meaningful fragmentation patterns [14–17]. Fast atom antimicrobials [2], free radical scavengers [1], metal bombardment (FAB) and liquid secondary ion mass chelators [1], and anti-viral and anti-bacterial agents [3]. spectrometry (LSIMS) have been used extensively [18– Since they are a ubiquitous part of the human diet, their 36]. In addition, thermospray [37–39] and atmospheric effect on human health is of interest. Flavonoids are pressure chemical ionization (APCI) [40, 41] have been known to have medicinal and chemopreventive activi- used for several applications involving the analysis of ties in humans [4–7]. For example, a diet rich in flavonoids. Most recent, electrospray ionization (ESI) flavonoids has shown to have an inverse relationship has become a popular choice [40–49]. For example, the with heart disease [8–10]. positive ESI mode was used to identify and quantitate There is a basic structure which is common to all flavonoids in soya flours and baby foods [43], and in flavonoids; however, there is a great diversity of fla- hops and beer [48]. Also, the negative ESI mode was vonoids due to different hydroxylation and glycosyla- used to identify and quantitate flavonoids in tea ex- tion positions. Most flavonoids exist as glycosides in tracts [44] and plants such as Passiflora incarnata [47]. plant sources, and many only differ by the nature of the Tandem mass spectrometry has been used to eluci- aglycone, and/or by the glycosylation site, the se- date many of these compounds [23, 24, 31–36, 44, 47, quence, and the interglycosidic linkages of the glycan 50–52]. For example, CAD of deprotonated flavonoid portion. The biological activities of flavonoids are af- glycosides commonly results in loss of the sugars (for- Ϫ Ϫ fected by these subtle structural differences [11]. There- mation of Y0 and Y1 ions). CAD of deprotonated fore, there is a critical need for the development of flavonoid-O-glycosides allows distinction of rutino- analytical methods to elucidate structurally similar sides (1 3 6 disaccharides) from neohesperidosides (1 compounds. 3 2 disaccharides) based on the greater abundance of Ϫ A number of mass spectrometric techniques have the Y1 ions for the neohesperidosides compared to the been used to study the structures of flavonoids [12]. rutinosides [50]. However, unique fragment ions were Electron ionization (EI) has been used to evaluate the not observed for each isomer in all cases, so some fragmentation pathways of some aglycones [13] and for identifications were based on differences in abundances limited structural studies of glycosides. However, der- of fragment ions of the same m/z values. In addition, CAD of protonated flavonoid-C-glycosides allowed the differentiation between 6-C and 8-C glycosides [51]. Published online October 30, 2003 However, the isomeric distinctions generally relied on Address reprint requests to Dr. J. S. Brodbelt, Department of Chemistry and Biochemistry, The University of Texas at Austin, 1 University Station the relative abundances of common product ions in the A5300, Austin, TX 78712-0165. E-mail: [email protected] MS/MS spectra for some compounds, and diagnostic © 2003 American Society for Mass Spectrometry. Published by Elsevier Inc. Received April 8, 2003 1044-0305/03/$30.00 Revised July 24, 2003 doi:10.1016/j.jasms.2003.07.002 Accepted July 25, 2003 1438 PIKULSKI AND BRODBELT J Am Soc Mass Spectrom 2003, 14, 1437–1453 Ϫ product ions were only observed in the MS/MS/MS the solutions was 5 L min 1. The lens and octapole spectra. Hvattum et al. used CAD to evaluate the voltages, sheath gas flow rate and capillary voltage formation of radical fragment ions from deprotonated were optimized for maximum intensity of the ion of flavonoid glycosides by loss of neutral sugar radicals interest. The capillary temperature was 200 °C. The [52]. The radical fragmentation processes were related interface pressure, measured with the convectron gauge to the number of hydroxyl substituents on the B ring of at the skimmer cone, was normally 0.9 torr. The pres- the flavonoid, in addition to the type and position of the sure in the ion trap with helium added was nominally Ϫ sugar substituent. It was surmised that the radical loss 1.9 ϫ 10 5 torr, measured by the ionization gauge. The of the 3™O-sugar substituent was enhanced by the spectra were acquired with ion injection times of 5 ms presence of multiple hydroxyl groups on the B ring due and an average of 10 microscans. to the electron-donating properties of the hydroxyl In the negative ESI mode, the ion corresponding to Ϫ groups, thus weakening the glycosidic bonds. [L-H] was optimized, where L is the flavonoid. In the Due to their acidic functional groups, protonation of positive ESI mode, when solutions of flavonoid/metal/ flavonoid glycosides is inefficient, often resulting in auxiliary ligand were used, the ion corresponding to ϩ weak positive ESI mass spectra. In some cases, the [M(II) (L-H) A] was optimized, where M is the metal fragmentation patterns of protonated flavonoids do not and A is the auxiliary ligand. During MS/MS experi- differentiate similar compounds or isomers. Although ments, these parent ions were isolated and the CAD deprotonation results in more intense mass spectra in voltage was adjusted so that the parent ion intensity the negative ESI mode, the fragmentation patterns decreased to 20% of the base peak. Ϫ again frequently do not differentiate similar com- Stock solutions of 4.0 ϫ 10 4 M metal salt/methanol Ϫ pounds or isomers. Metal complexation is an alternative and 4.0 ϫ 10 4 M auxiliary ligand/methanol were used ionization mode which has been explored in the Brod- to create the analytical solutions. Solutions containing a belt group in the past [53–58]. It has been observed that flavonoid, a metal salt, and an auxiliary ligand were Ϫ metal complexation can both increase ion intensity and ϳ1:1:1 at 1.0 ϫ 10 5 M. The analytical solutions used for Ϫ alter fragmentation pathways, resulting in many more the negative ESI experiments were 1.0 ϫ 10 5 M fla- structurally distinctive fragment ions. Dramatic in- vonoid in methanol. creases in intensities of some flavonoids [56] have been The flavonoids apigenin, vitexin, apigenin-7-glu- observed with the use of copper, nickel, or cobalt, along coside, isorhoifolin, rhoifolin, isovitexin, quercetin, lu- with 2,2Ј-bipyridine or 1,10-phenanthroline as an aux- teolin, quercitrin, kaempferol-3-glucoside, luteolin-4Ј- iliary ligand. In this study, we show differentiation of glucoside, luteolin-7-glucoside and orientin were three series of flavonoid glycosides with the use of purchased from Indofine (Somerville, NJ). 2,2Ј-Bipyri- 2,2Ј-bipyridine (bpy) and 4,7-diphenyl-1,10-phenanth- dine, 4,7-diphenyl-1,10-phenanthroline, 1,10-phenanth- roline (dpphen), a new auxiliary ligand. Although some roline, 2,2Ј:6Ј,2Љ-terpyridine, 4,4Ј-dimethyl-2,2Ј-bipyri- of these compounds have been differentiated in the dine, CoBr2, NiBr2, and CuBr2 were purchased from positive ion mode based on differences in the relative Aldrich (Milwaukee, WI). The HPLC grade methanol intensities of specific fragment ions [50, 51] and in the was purchased from EM Science (Gibbstown, NJ). All negative ion mode based on low intensity fragment ions materials were used without further purification. formed upon CAD [51], in this work we are able to The nomenclature for glycoconjugates proposed by differentiate isomers via more intense, distinctive ions Domon and Costello [67] is used to describe the frag- in the CAD spectra. mentation pathways for O-glycosides. Fragments from g,h Two classes of flavonoids, flavones and flavonols, a terminal sugar unit are labeled using Ai,Bi and Ci, are considered in this study. In the present report, we where i is the number of the bond broken, counting extend and expand the use of metal complexation for from the terminal sugar beginning with 1, and g and h differentiation within these two classes of flavonoids. are the cross-ring cleavages of the sugar. Fragments k,l We accomplish differentiation of glycosides of api- which include the aglycone are labeled Xj,Yj, and Zj, genins, commonly found in olive oil [59], orange juice where j is the number of the bond broken, counting [60], celery [61], and garlic [62] (Figure 1, Group I); in from the aglycone beginning with 0, and k and l are the addition to glycosides of kaempferol, commonly found cross-ring cleavages of the sugar.