Silver Complexation and Tandem Mass Spectrometry for Differentiation of Isomeric Flavonoid...

Silver Complexation and Tandem MassSpectrometry for Differentiation of IsomericFlavonoid Diglycosides

Junmei Zhang and Jennifer S. Brodbelt*

Department of Chemistry and Biochemistry, The University of Texas, Austin, Texas 78712

For detection and differentiation of isomeric flavonoids,electrospray ionization mass spectrometry is used togenerate silver complexes of the type (Ag + flavonoid)+.Collisionally activated dissociation (CAD) of the resulting1:1 silver/flavonoid complexes allows isomer differentia-tion of flavonoids. Eighteen flavonoid diglycosides con-stituting seven isomeric series are distinguishable fromeach other based on the CAD patterns of their silvercomplexes. Characteristic dissociation pathways allowidentification of the site of glycosylation, the type ofdisaccharide (rutinose versus neohesperidose), and thetype of aglycon (flavonol versus flavone versus flavanone).This silver complexation method is more universal thanprevious metal complexation methods, as intense silvercomplexes are observed even for flavonoids that lack thetypical metal chelation sites. To demonstrate the feasibilityof using silver complexation and tandem mass spectrom-etry to characterize flavonoids in complex mixtures,flavonoids extracted from grapefruit juice are separatedby high-performance liquid chromatography and analyzedvia a postcolumn complexation ESI-MS/MS strategy.Diagnostic fragmentation pathways of the silver complexesof the individual eluting flavonoids allow successfulidentification of the six flavonoids in the extract.

The impact of diet on human health has become an active areaof research in recent years due to the growing recognition of thenumerous chemopreventive properties of naturally occurringcompounds, such as flavonoids,1-3 in fruits and vegetables. Thereare thousands of different flavonoids,1-3 with some possessing fargreater biological activities than others, and thus, the mapping ofstructure/activity relationships is an important goal, as well asthe development of versatile analytical methods for enhanceddetection and structural characterization of trace quantities offlavonoids and biotransformation products in food sources, urine,plasma, and tissue. Tandem mass spectrometry has been particu-

larly effective for the structural characterization of flavonoids.4-25

In the past four years, our group has focused considerableattention on developing novel mass spectrometric ways to detectand differentiate isomeric flavonoids based on metal complexation,including the use of transition metals,20-24 with20-23 or without24

an auxiliary ligand, alkaline earth metals,24 and aluminum.25 Metalcomplexation alters the fragmentation pathways of flavonoids,which allows differentiation of isomeric flavonoids in a systematicfashion.20-25 Though success has been achieved, one limitationof the above metal complexation approaches is that flavonoidsmust have a 4-keto group and at least one neighboring hydroxylgroup for formation of strong complexes with the metals.Therefore, an alternative method is needed for flavonoids that donot have such structural features. In this work, we report the useof silver(I) as a metal complexation reagent for electrosprayionization of flavonoids followed by tandem mass spectrometry.

* Corresponding author. Phone: (512) 471-0028. Fax: (512) 471-8696.E-mail: [email protected].(1) Bohm, B. A. Introduction to Flavonoids; Harwood Academic Publishers:

Singapore, 1998; Chapter 2.(2) Middleton, E., Jr.; Kandaswami, C. In The Flavonoids: Advances in Research

since 1986; Harborne J. B., Ed.; Chapman & Hall: London, 1994; Chapter15.

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10.1021/ac048818g CCC: $30.25 © 2005 American Chemical Society Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1761Published on Web 02/04/2005

Silver complexation has been used for effective ionization ofseveral classes of compounds in both liquid secondary ion massspectrometry and electrospray ionization applications to enhancethe ionization efficiencies of the compounds, improve the selectiv-ity and sensitivity of the mass spectrometric methods, or alterthe fragmentation patterns of the molecular ions.26-51 Within thepast decade, silver complexation has been applied to a variety ofcompounds that are nonpolar and not easily ionizable, such asmethyl glycosides,26 fatty acid methyl esters,27 tocopherols andcarotenoids,28 olefins, polyolefins and aromatic compounds,29 diacylperoxides,30 barbiturates and chlorinated alkyl phenoxypro-panoates,31 heavy aromatic petroleum fractions,32 monosaccha-rides,33 polyaromatic hydrocarbons,34 peroxidation products ofcholesterols such as phospholipids and docosahexaenoate esterhydroperoxides,35-38 and rubber vulcanization products.39 In ad-dition to enhanced isomer differentiation or improved selectivityand sensitivity for some of the previous work, silver complexationenables ionization of nonpolar compounds. Silver(I) can either bemixed directly with compounds of interest or be introduced as apostcolumn complexation reagent after the compounds are

separated by high-performance liquid chromatography (HPLC).Silver(I) also has been investigated as a complexation reagent foramino acids, peptides, and proteins by several groups in the pastfew years.40-51 Argentinated (silver-containing) peptides and aminoacids have altered fragmentation pathways (compared to theprotonated species) that can be useful for sequencing.

In the current work, the formation of silver/flavonoid com-plexes by electrospray ionization and differentiation of isomersby tandem mass spectrometry is reported for flavonoid glycosidesand aglycons. In general, abundant 1:1 silver(I)/flavonoid com-plexes of the type (Ag + flavonoid)+ are observed. Collisionallyactivated dissociation (CAD) is applied for the structural charac-terization of isomeric flavonoid diglycosides after silver complex-ation. Unique fragments of high abundances are used to differ-entiate the flavonoids in each isomeric series. The structuralfeatures that lead to specific fragmentation patterns are alsodiscussed in detail. The feasibility of the method for LC/MSapplications via a postcolumn complexation strategy is demon-strated for the separation and identification of six flavonoids (threegroups of isomers) in a grapefruit juice extract.

EXPERIMENTAL SECTIONChemical Reagents. (+)-Catechin hydrate, daidzein, hespe-

ridin, naringin, neohesperidin, and rutin were purchased fromSigma (St. Louis, MO). Diosmin, isorhoifolin, narirutin, neo-diosmin, and rhoifolin were purchased from Indofine (Somerville,NJ). Daidzin, datiscoside, didymin, eriocitrin, fortunellin, kaempferol-7-O-neohesperidoside, kaempferol-3-O-rutinoside, linarin, neoerio-citrin, and poncirin were purchased from Extrasynthese (Genay,France). Biochanin A was purchased from Aldrich (Milwaukee,WI). Silver nitrate was purchased from Johnson Matthey (WardHill, MA). Formic acid was from EM Science (Gibbstown, NJ).All the above compounds were used without further purification.All the solvents were HPLC grade. The structures of all theflavonoid diglycosides are listed in Figure 1 and those of theaglycons and monosaccharide (daidzin) are shown in Figure 2.All the stock solutions of flavonoids (10-4-10-3 M) and silvernitrate (10-3-10-2 M), as well as the working solutions offlavonoids and silver/flavonoid complexes, were prepared in HPLCgrade methanol.

Direct Infusion. A Thermo Finnigan LCQ Duo quadrupoleion trap instrument equipped with an electrospray ionization (ESI)source (San Jose, CA) was used. The flow rate of the silver(I)/flavonoid solutions was 5 µL/min. The ESI spray voltage was +5.0kV. The heated capillary temperature was 210 °C. The flow rateof the sheath gas (nitrogen) was 20 arbitrary units, and auxiliarygas was not used. The injection time (i.e., ionization time) was10-50 ms. The other instrumental parameters were tuned tooptimize the relative abundance of a typical complex (Ag + rutin)+.An isolation window of 1.5 m/z was used for all the CADexperiments. The CAD energy (mass corrected, % of 5 V0-p) wasvaried such that only 5-15% of a parent ion survived the process.Each spectrum was an average of 10 scans, and each scan wasan average of 10 microscans. The isomers in each isomeric serieswere run back-to-back on the same day. When the CAD patternsof deprotonated or sodium-cationized flavonoids were of interest,the flavonoids were introduced to the LCQ without mixing withsilver nitrate. Very similar experimental conditions were used

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1762 Analytical Chemistry, Vol. 77, No. 6, March 15, 2005

except that the ESI spray voltage was switched to -4.5 kV fordeprotonation.

LC/MS with Postcolumn Complexation. Flavonoids wereextracted from a commercial grapefruit juice sample as describedby Zhang and Brodbelt.52 The flavonoids in the extract wereseparated using a Waters Alliance 2690 system (Waters, Milford,

MA). A Waters Symmetry C18 column (2.1 × 50 mm, 3.5 µm)was used with a guard (2.1 × 10 mm, 3.5 µm). The mobile phasewas composed of solvents A (0.33% formic acid), B (acetonitrilewith 0.33% formic acid), and C (2-propanol with 0.33% formic acid).

(52) Zhang, J.; Brodbelt, J. S. Analyst 2004, 129, 1227-1233.

Figure 1. Flavonoids studied for isomer differentiation (molecular weight, class of flavonoid).

Analytical Chemistry, Vol. 77, No. 6, March 15, 2005 1763

A gradient method was used to separate the flavonoids. SolventC was kept at 7% throughout the entire run. For the first 6 min,solvent B was increased from 10 to 28% and then brought back to10% over the next 2 min. The initial conditions were held for 12min prior to the next injection. The injection volume was 5 µL.The flow rate was 100 µL/min. The HPLC effluent was mixedwith 1 × 10-3 M silver nitrate (introduced by a syringe pump at20 µL/min) via a tee before entering the LCQ.

For LC/MS analysis, the automated gain control option wasengaged, and selected ion monitoring was used. The flow rate ofthe sheath gas was 50 arbitrary units and that of the auxiliary gas(nitrogen) was 20 arbitrary units. The heated capillary temperaturewas raised to 250 °C, and the other parameters were the same asfor the direct infusion experiments. The maximum injection timewas set at 2000 ms with 2-microscan averaging. The ionsmonitored were the silver complexes of narirutin and naringin(m/z 687), hesperidin and neohesperidin (m/z 717), and didyminand poncirin (m/z 701).

For acquisition of the fragmentation patterns of the silvercomplexes in the LC/MS/MS experiments, the maximum injec-tion time was 200 ms with 5 microscans. The isolation windowwas set at 1.5 m/z. The CAD voltage, expressed as percentage of5 V0-p, was 33% (where 100% is 5 V0-p) for sufficient dissociationof all the complexes.

RESULTS AND DISCUSSIONFlavonoids usually give higher signals upon deprotonation in

the negative mode than protonation or sodium cationization inthe positive mode because of their acidic hydroxyl groups.However, deprotonated flavonoids tend to undergo very simpleCAD dissociation pathways, dominated by the loss of the saccha-ride portion. For example, deprotonated datiscoside dissociatesalmost exclusively by the loss of the disaccharide group (m/z 285)(Figure 3A). The CAD patterns of sodium-cationized flavonoidsare in general more complex, which include several fragmentationpathways as demonstrated by that of datiscoside (Figure 3B).Sodium-cationized datiscoside dissociates by the losses of theaglycon residue (m/z 331), the rhamnose moiety (m/z 471), andthe disaccharide group (m/z 309) as well as dehydration (m/z599). Despite the richer arrays of dissociation pathways of thesodium-cationized flavonoids, sodium cationization fails to serveas a general strategy for flavonoid characterization due to the very

low (if any) abundances of the sodium complexes. Therefore,alternative methods are needed for more effective characterizationof structurally similar flavonoids.

Silver(I) forms strong complexes (their intensities are similarto or higher than the corresponding deprotonated flavonoids andmuch higher than the sodium-cationized flavonoids) of the type(Ag + L)+ with flavonoids, where L is the flavonoid. A typical ESIfull-scan spectrum obtained from a mixture of rutin and silvernitrate is shown in Figure 4. The 1:1 silver/rutin complexdominates the mass spectrum, and this 1:1 stoichiometry isconsistently favored for all the flavonoids with minimal competingproducts. In contrast to the simple fragmentation patterns ofdeprotonated flavonoids,13,20-25 the silver/flavonoid complexesprovide richer arrays of dissociation pathways and more diagnosticproduct ions for flavonoid characterization. For example, the silvercomplex of datiscoside dissociates by the losses of the aglyconresidue (m/z 415), the aglycon residue and one water molecule(m/z 397), the aglycon plus 64 Da (m/z 351), the aglycon plusthe rhamnose moiety and one water molecule (m/z 251), and therhamnose moiety (m/z 555) (Figure 3C).

In the following sections, the conditions that influence forma-tion of the silver complexes are investigated and optimized, andthe ability to differentiate seven series of isomeric flavonoid

Figure 2. Additional flavonoids screened for silver complexation(molecular weight, class of flavonoid).

Figure 3. CAD spectra of datiscoside after deprotonation, sodiumand silver complexation. The parent ions are labeled with asterisks.Losses: R ) rhamnose residue (146), A ) aglycon moiety, and D )disaccharide group (308).

Figure 4. Representative ESI full scan spectrum of a mixture ofrutin (1 × 10-5 M) and silver nitrate (5 × 10-5 M) in methanol.


diglycosides by CAD is evaluated. As a demonstration of thefeasibility of using this approach for characterizing flavonoids incomplex mixtures, flavonoids extracted from grapefruit juice wereseparated by HPLC and then characterized by postcolumn silvercomplexation and tandem mass spectrometry.

Investigation of Complexation Conditions. 1. Optimizationof the Concentration of Silver Nitrate. Rutin was used as amodel compound to investigate the influence of the silver nitrateconcentration on the relative signal intensities of the complexesacross a broad concentration range. The concentration of rutinwas kept at 1 × 10-5 M while the concentration of silver nitratewas varied from 2 × 10-6 to 1 × 10-4 M. It can be seen fromTable 1 that, as the concentration of silver nitrate increases, sodoes the complex intensity until the signal levels off when theconcentration ratio reaches ∼1:5 (rutin/silver nitrate). Therefore,the 1:5 flavonoid/silver nitrate concentration ratio was used forall the other flavonoids of interest.

2. Structural Requirement of Flavonoids. One of the long-term objectives of our flavonoid work is to find a metal thatpromotes efficient ionization of all flavonoids, even those that donot have both a 4-keto group and at least one neighboringhydroxyl group as required for many other metal coordinationstrategies,20,24,25 to provide a more universal approach. To gaininsight into the structural requirements of flavonoids for silvercomplexation by ESI-MS, a series of flavonoids were screenedfor their silver complexation abilities. Besides the flavonoiddiglycosides listed in Figure 1, which all have a 4-keto group anda 5-OH group, several flavonoid aglycons and a monoglycoside(daidzin) that do not have the above structural features were alsotested (Figure 2).

(+)-Catechin is a flavonoid that does not have a 4-keto group,but the intensity of its silver complex is as high as that of biochaninA, which indicates that a 4-keto group is not essential for silvercomplexation. Daidzein and daidzin are two flavonoids that havethe 4-keto group but lack a neighboring hydroxyl group (3- or5-OH). The formation of abundant 1:1 complexes of theseflavonoids with silver(I) suggests that the coexistence of a 4-ketogroup and at least one adjacent hydroxyl group is not requiredfor silver complexation.

Isomer Differentiation of Flavonoid Diglycosides. Oneunique feature of the silver(I) ion is that it has two isotopes (107and 109 Da) with almost equal abundance. Therefore, the resultingESI mass spectra for the 1:1 silver/flavonoid complexes have twoadjacent peaks: (107Ag + L)+ and (109Ag + L)+ with similarintensities as shown in Figure 4. The presence of two isotopicpeaks provides a convenient way to determine whether thefragment ions in the CAD mass spectra retain the silver ion ornot based on comparison of m/z values for the fragment ionsproduced from each isotopic parent ion. Selective isolation and

activation of each of the two parent ions leads to a series offragment ions (Figure 5). If the m/z values are the same for apair of such fragments, the fragments do not contain the silverion. If the m/z values differ by 2 Da, then the pair of fragmentsretains the silver ion. Most of the fragment ions in this studyinvolve retention of the silver ion. However, there are a few minorfragments that do not contain the silver ion. For example, them/z 301 ions for the hesperidin and neohesperidin complexes,the m/z 287 ions for the eriocitrin and neoeriocitrin complexes,and the m/z 285 ions for the didymin and poncirin complexes allinvolve the loss of silver, the disaccharide group, and a hydrogenatom from the corresponding parent ions. Because these ionsoccur with such low intensities (less than 10% intensity) and arenot highly diagnostic, they are not tabulated. For simplicity, onlythe CAD data obtained from the (107Ag + L)+ complexes arediscussed below.

It has been challenging to differentiate isomeric flavonoidsbecause the structural differences among isomers are often subtle,involving only the inter-saccharide linkage or occasionally theglycosylation site or the type of aglycon as shown in Figure 1.According to their molecular weights, the flavonoid diglycosidesin Figure 1 are divided into seven isomeric series (MW 610, 608,596, 594, 592, 580, and 578). Two sets of representative CADspectra for the MW 580 and 578 isomers are shown in Figures 6and 7. The major CAD product ions of all the flavonoid complexesare summarized in Table 2. The silver complexes of structurallysimilar flavonoids have CAD fragmentation pathways distinct fromeach other, which are used for structural characterization andisomer differentiation as discussed below. Some of the majordissociation pathways are illustrated in Scheme 1 for the silvercomplex of rutin. The correlations between the structural featuresand CAD fragmentation patterns of the flavonoids are alsodiscussed in detail.

The 3-rutinosides have a richer array of fragments than the7-rutinosides and 7-neohesperidosides, and thus, rutin serves asa good model for illustrating the diagnostic fragmentation path-ways of the silver complexes. The five major fragmentationpathways include the losses of the rhamnose residue, the aglyconmoiety, the aglycon moiety in conjunction with dehydration, theaglycon group plus the loss of 64 Da, and the aglycon group along

Table 1. Relative Intensity of the (Ag + Rutin)+

Complex in Methanol

concn ratio (rutin/AgNO3)a 5:1 2:1 1:1 1:2 1:5 1:10complex intensityb 0.024 0.21 0.26 0.74 1.0 0.95

a The concentration of rutin was kept at 1 × 10-5 M. The solventused was 100% methanol. b All the complex signals were compared tothe one obtained when the concentration ratio was 1:5.

Figure 5. CAD spectra of the (Ag + rutin)+ complex. The parentcomplex ions are labeled with asterisks. Losses: R ) rhamnoseresidue (146) and A ) aglycon moiety.


with the rhamnose residue and water (confirmed by MSn) asshown in Scheme 1. The elimination of 64 Da mentioned abovemay occur via cleavage across one or both of the saccharides andinvolve fast consecutive losses (i.e., the loss of water and 46 Da(a C2H6O moiety or H2O and CO) from the saccharide residues,as confirmed by MSn experiments. The actual coordination sitesof the silver ion are not known for either the parent or the resultingfragment ions. There may be more than one structure for aparticular ion, but only one is shown in Scheme 1 for simplicity.For example, dehydration may occur on either of the twosaccharide rings, and it is impossible to pinpoint where the waterloss occurs with any certainty.

1. Isomer Differentiation. MW 610 Isomers. Hesperidin andneohesperidin are two 7-O-diglycosides of the same flavanoneaglycon hesperetin. They differ only by one structural feature: theinter-saccharide linkage. Hesperidin has a 1-6 rhamnose-glucoselinkage (rutinoside), while neohesperidin has a 1-2 rhamnose-glucose linkage (neohesperidoside). Despite this subtle structuraldifference, the silver complexes of these two flavonoids have

distinct CAD patterns (Table 2). For example, the dominantdissociation pathway of the hesperidin complex is the loss of therhamnose residue (m/z 571) along with two minor fragmentationpathways (each with less than 15% relative abundance). There isa second major fragmentation pathway for the neohesperidincomplex: the loss of the aglycon moiety (m/z 415).

The third isomer in this series is rutin, a 3-O-rutinoside ofquercetin, a flavonol. For the silver complex, the primary dis-sociation pathway is the elimination of the aglycon moiety (m/z415) instead of the loss of the rhamnose residue that is thedominant fragmentation pathway of the complexes of the othertwo isomers (Figure 5 and Table 2). The rutin complex yieldsthree additional key fragments: the loss of the aglycon moiety inconjunction with dehydration (m/z 397), the loss of the aglyconresidue plus 64 Da (the sugar rearrangement and cleavageprocess) (m/z 351), and the loss of both the aglycon and therhamnose moieties plus one water molecule (m/z 251).

MW 608 Isomers. As two 7-O-diglycosides of diosmetin (aflavone), diosmin and neodiosmin differ from hesperidin andneohesperidin, respectively, only by the bond character betweenC2 and C3 of their aglycons. Diosmetin has a double bond,whereas hesperetin has a single bond. The silver complexes ofdiosmin and neodiosmin produce significantly different CADspectra. The diosmin complex gives only one product ion via theloss of the rhamnose residue (m/z 569) upon CAD. The dominantproduct of the neodiosmin complex is also the elimination of therhamnose moiety (m/z 569), but it has two additional fragmentsincluding the loss of the aglycon residue (m/z 415) and the lossof the rhamnose group plus one water molecule (m/z 551) (Table2).

MW 596 Isomers. The second series of flavanone isomersinclude eriocitrin and neoericitrin, two diglycosides of eriodictyol.Compared to hesperidin and neohesperidin, the substituent at the4′-position of these two flavonoids is a hydroxyl instead of amethoxy group. Not surprisingly, the silver complexes of thesetwo series of isomers have similar CAD patterns, which aregoverned by the inter-saccharide linkage rather than the aglyconstructure (for the same type of aglycon). Loss of the rhamnoseresidue (m/z 557) is dominant for both of the isomeric complexes,in addition to the loss of the disaccharide moiety (m/z 395) (Table2). The neoeriocitrin complex has the additional loss of theaglycon moiety (m/z 415), elimination of the aglycon and therhamnose residues (m/z 269), and the dehydration product of thelatter ion (m/z 251).

MW 594 Isomers. This isomeric series includes five fla-vonoids: datiscoside (datiscetin-3-O-rutinoside), kaempferol-3-O-rutinoside, kaempferol-7-O-neohesperidoside, didymin (isos-akuranetin-7-O-rutinoside), and poncirin (isosakuranetin-7-O-neohesperidoside). The only difference between datiscetin andkaempferol is the position of the single hydroxyl group on the Bring (2′- vs 4′-). Isosakuranetin is a flavanone aglycon similar tohesperetin, while datiscetin and kaempferol are flavonols. Thesilver complexes of the two 3-O-rutinosides (datiscoside andkaempferol-3-O-rutinoside) share the same fragment ions, includ-ing losses of the aglycon residue (m/z 415), the rhamnose moiety(m/z 555), the aglycon residue plus one water molecule (m/z 397),the aglycon residue plus 64 Da (the sugar rearrangement andcleavage reaction) (m/z 351), the disaccharide group with addition

Figure 6. CAD spectra of the (107Ag + L)+ complexes of flavonoidswith MW 580. The parent complex ions are labeled with asterisks.Losses: R ) rhamnose residue (146), A ) aglycon moiety, and D )disaccharide group (308).

Figure 7. CAD spectra of the (107Ag + L)+ complexes of twoflavonoids with MW 578. The parent complex ions are labeled withasterisks. Losses: R ) rhamnose residue (146), A ) aglycon moiety,and D ) disaccharide group (308).


of one water molecule (m/z 411), and a few ions in the low-massrange (m/z 251, 253, 269, etc.) (Table 2). Despite the similaritybetween datiscoside and kaempferol-3-O-rutinoside, these two

flavonoids are distinguishable from each other by the significantdifferences in the relative intensities of product ions at m/z 555,397, and 351, in addition to the low-mass fragment ions. The CAD

Table 2. Major CAD Product Ions of the (107Ag + L)+ Complexes of Flavonoids

CAD product ionsb,c (m/z, (%))

flavonoid parenta - R- (R +H2O) - A

- (A +H2O)

- D +H2O - D

- (A +64)d

- (A +R)

- (A +R + H2O) others

hesperidin 717 (15) 571 (100) 415 (11) 409 (14)neohesperidin 717 (13) 571 (100) 415 (79) 409 (22)rutin 717 (10) 571 (31) 415 (100) 397 (29) 351 (25) 251 (12)diosmin 715 (11) 569 (100)neodiosmin 715 (10) 569 (100) 551 (10) 415 (45)eriocitrin 703 (8) 557 (100) 395 (32)neoeriocitrin 703 (9) 557 (100) 415 (62) 395 (38) 269 (12) 251 (10)datiscoside 701 (9) 555 (24) 415 (100) 397 (29) 411 (10) 351 (26) 251 (13) 253 (10)kaempferol-3-O-

rutinoside701 (11) 555 (71) 415 (100) 397 (42) 411 (15) 351 (44) 269 (13) 251 (25) 271 (11)

253 (20)kaempferol-7-O-

neohesperidoside701 (13) 555 (100) 537 (23) 415 (45) 411 (11)

didymin 701 (12) 555 (100) 415 (14) 393 (17)poncirin 701 (8) 555 (100) 415 (66) 393 (40)linarin 699 (11) 553 (100)fortunellin 699 (11) 553 (100) 535 (14) 415 (38) 409 (11) 251 (10)narirutin 687 (10) 541 (100) 415 (12) 379 (26)naringin 687 (10) 541 (100) 415 (75) 379 (28) 269 (11) 251 (12)isorhoifolin 685 (8) 539 (100)rhoifolin 685 (11) 539 (100) 521 (16) 415 (42) 395 (16) 251 (11)

a The m/z of the parent complex and its relative intensity after CAD are given. b Only fragment ions with more than 10% relative intensities arelisted. c The losses are abbreviated as follows: R ) rhamnose residue (146), A ) aglycon moiety, and D ) disaccharide group (308). d The eliminationof 64 Da may involve consecutive losses, i.e., the loss of water and 46 Da (a C2H6O moiety or H2O and CO).

Scheme 1. CAD Fragmentation Pathways for the (Ag + Rutin)+ Complex Using Speculative Structuresof the Fragment Ionsa

a Only the m/z values corresponding to one isotope of the silver ion (107) are listed for simplicity. The letters A and R represent theaglycon and rhamnose residues, respectively. The elimination of 64 Da may involve consecutive losses, i.e., the loss of water and 46Da (a C2H6O moiety or H2O and CO). Please note that the dashed lines in the proposed structures indicate that the actual coordinationsites of the silver ion are not known.


pattern of the kaempferol-7-O-neohesperidoside complex is simplerand quite different from the above two flavonol complexes byhaving the loss of the rhamnose residue (m/z 555) (instead ofthe loss of the aglycon residue (m/z 415)) as the most dominantfragmentation pathway, and the other fragmentation pathwaysshared by the datiscoside and kaempferol-3-O-rutinoside com-plexes are much less pronounced for the kaempferol-7-O-neohes-peridoside complex. In addition, the silver complex of kaempferol-7-O-neohesperidoside has a unique fragment ion at m/z 537,stemming from the loss of the rhamnose residue plus one watermolecule (Table 2).

Similar to the complex of kaempferol-7-O-neohesperidoside,the CAD mass spectra of the complexes of the two flavanonediglycosides, didymin and poncirin (also with MW 594), aredominated by the loss of the rhamnose residue (m/z 555) (Table2). These two flavanones can be distinguished from kaempferol-7-O-neohesperidoside by the presence of an additional fragmention at m/z 393 (from the loss of the disaccharide residue) andthe absence of the m/z 537 ion (loss of the rhamnose group andone water molecule). The CAD pattern of the poncirin complexdiffers from the didymin complex by its much more pronouncedfragments at m/z 415 (from the loss of the aglycon moiety) and393. The dissociation patterns of the silver complexes allowssuccessful differentiation of the five isomeric flavonoids, whichdemonstrates the advantage of silver complexation over de-protonation or transition metal complexation.21-23

MW 592 Isomers. The second flavone isomer series includeslinarin and fortunellin, which are two acacetin 7-O-diglycosides.Similar to the complexes of the other flavone 7-rutinosides, the(linarin + Ag)+ complex dissociates exclusively via the loss ofthe rhamnose residue (m/z 553). The corresponding fortunellincomplex yields four other fragment ions, one due to the loss ofthe aglycon moiety (m/z 415), one due to the loss of the rhamnoseresidue plus one water molecule (m/z 535), one due to the lossof the disaccharide moiety with rapid addition of a water molecule(m/z 409), and one involving elimination of both the aglycon andrhamnose residues plus loss of one water molecule (m/z 251)(Table 2).

MW 580 Isomers. As the fourth flavanone pair, narirutin andnaringin are two 7-O-diglycosides of naringenin. The CAD patternsof the silver complexes allow differentiation of these two flavonoids(Figure 6 and Table 2). Both complexes share the same dominantloss of the rhamnose residue (m/z 541) followed by the losses ofthe aglycon (m/z 415) and the disaccharide (m/z 379) residues,yet the product ion from the loss of the aglycon group is six timesmore intense for the naringin complex than for the narirutincomplex. In addition, the naringin complex has more intensefragment ions at m/z 269 and 251 (stemming from the losses ofthe aglycon and rhamnose residues without and with a secondarydehydration reaction).

MW 578 Isomers. Isorhoifolin and rhoifolin, two 7-O-diglyco-sides of apigenin, are the flavone versions of narirutin andnaringin, respectively. The isorhoifolin complex only dissociatesvia the loss of the rhamnose moiety (m/z 539). In contrast, thereare four additional pathways for the rhoifolin complex: loss ofthe aglycon moiety (m/z 415), loss of the rhamnose residue andone water molecule (m/z 521), loss of the disaccharide withaddition of one water molecule (m/z 395), and elimination of both

the aglycon and rhamnose residues in conjunction with dehydra-tion (m/z 251) (Figure 7 and Table 2).

2. Correlations between Structural Features and CADFragmentation. The CAD fragmentation pathways cannot onlybe used to differentiate isomeric flavonoids, but they correlatewith the specific classes of flavonoids, with the latter moreimportant for characterizing newly discovered flavonoids. Forexample, the three flavones that are 7-O-rutinosides (diosmin,linarin, isorhoifolin) undergo only one dominant dissociationreaction: loss of the rhamnose residue. The four flavanones thatare 7-O-rutinosides (hesperidin, eriocitrin, didymin, narirutin)undergo at least one or two additional fragmentation pathwaysbesides the dominant loss of the rhamnose residue: loss of thedisaccharide group and loss of the aglycon moiety. In contrast,the loss of the aglycon residue becomes dominant for the three3-O-rutinosides (rutin, datiscoside, kaempferol-3-O-rutinoside)along with at least four other dissociation routes including lossof the rhamnose residue, loss of the aglycon moiety plus one watermolecule, loss of the aglycon plus 64 Da (the sugar rearrangementand cleavage process), and elimination of the aglycon andrhamnose groups in conjunction with dehydration. All of the silvercomplexes containing flavonoid 7-O-neohesperidosides share thesame dominant loss of the rhamnose residue with their 7-O-rutinoside isomers, but the neohesperidoside complexes eitherhave additional fragmentation pathways or the intensities of thefragment ions other than the loss of the rhamnose residue aremuch higher than the corresponding rutinoside complexes. Forexample, the loss of the aglycon residue is over four times morepronounced for a neohesperidoside complex than for the corre-sponding rutinoside counterpart. Therefore, the CAD fragmenta-tion patterns of the silver complexes can be used to assist inclassifying a flavonoid as either a rutinoside or a neohesperidoside,as either 7-O or 3-O glycosylated, and as either a flavanone or aflavone or a flavonol.

LC/MS with Postcolumn Complexation. A good isomerdifferentiation method should not only work well for purifiedcompounds but should also work well for compounds in complexmixtures. Several flavonoids, including isomeric ones, coexist incitrus products, i.e., naringin, narirutin, hesperidin, neohesperidin,didymin, and poncirin in some brands of grapefruit juice,53 andrutin, hesperidin, neohesperidin, naringin, narirutin, didymin, andponcirin in citrus leaves54 and citrus fruits.55 Therefore, identifica-tion of flavonoids including isomers in mixtures is an importantanalytical task. As an application of using silver complexation andtandem mass spectrometry for differentiating isomeric flavonoids,flavonoids extracted from grapefruit juice were analyzed basedon a postcolumn complexation LC/MS strategy. Silver complexesfor the eluting flavonoids were formed by introducing the silvernitrate reagent in excess via a tee to the HPLC effluent. Theresulting silver complexes were then detected by ESI-MS withcharacterization by tandem mass spectrometry.

A typical total ion chromatogram is shown in Figure 8A. Todeconvolute the chromatogram and detect minor flavonoids in

(53) Ross, S. A.; Ziska, D. S.; Zhao, K.; Elsohly, M. A. Fitoterapia 2000, 71,154-161.

(54) Kawaii, S.; Tomono, Y.; Katase, E.; Ogawa, K.; Yano, M.; Koizumi, M.; Ito,C.; Furukawa, H. J. Agric. Food Chem. 2000, 48, 3865-3871.

(55) Kawaii, S.; Tomono, Y.; Katase, E.; Ogawa, K.; Yano, M. J. Agric. Food Chem.1999, 47, 3565-3571.


the complex extract, selected ion chromatograms are recon-structed as shown in Figure 8B-D. It can be seen that two majorisomeric flavonoids (retention times (RT) ) 8.4, 10.0 min) existin the grapefruit juice studied, which have a molecular mass of580 Da (i.e., 687-107 (Ag)). Besides the two major flavonoids,four minor flavonoids belonging to two isomeric series are alsodetectable in the extract: MW 594 (i.e., 701-107 (Ag)) with RT15.7 and 17.0 min and MW 610 (i.e., 717-107 (Ag)) with RT 9.4and 11.1 min. It can also be seen that good separation wasachieved for each of the three isomeric series of flavonoids usingthe C18 column under the gradient conditions as described in theExperimental Section.

To pinpoint the identity of each of the eluting flavonoids, CADwas used to probe the complexes. The corresponding CAD spectrafor the six flavonoids with different retention times are shown inFigure 9. The two major flavonoids dissociate via the dominantloss of the rhamnose residue (m/z 541) and by the loss of thedisaccharide (m/z 379) or the aglycon (m/z 415) residues (Figure9A,B), which suggests that they are two flavanones. The fact thatthe loss of the aglycon group (m/z 415 fragment ion) is muchmore intense for the second eluting flavonoid (RT ) 10.0 min)than the first (RT ) 8.4 min) confirms that the second elutingflavonoid is naringin and the first is narirutin. The four minorflavonoids have similar CAD patterns to the above two flavanones,respectively, with the loss of the aglycon residue more pronouncedfor the flavonoids with longer retention times than their isomers(Figure 9C-F). Therefore, the four minor flavonoids are didymin(RT ) 15.7 min), poncirin (RT ) 17.0 min), hesperidin (RT ) 9.4min), and neohesperidin (RT ) 11.1 min). The flavonoidsidentified are in accordance with the ones reported.53 This exampledemonstrates that postcolumn silver complexation can be usedwith tandem mass spectrometry to successfully identify flavonoidsin mixtures.

CONCLUSIONSSilver complexation in an ESI-MS/MS strategy allows en-

hanced isomer differentiation of flavonoids. This metal complex-ation method is robust and more universal than previous metalcomplexation strategies that involved divalent transition metalswith auxiliary ligands, trivalent aluminum, or alkaline earth metals.Unlike the previous metal complexation methods, the presenceof a 4-keto group and at least one neighboring hydroxyl group(3-, 5-, or both) is not essential for the formation of strong silvercomplexes. The CAD patterns of the resulting 1:1 silver(I)/flavonoid complexes are useful for structural characterization andisomer differentiation of seven series of isomeric flavonoiddiglycosides. In addition to distinguishing isomeric flavonoids,correlations are identified between the CAD fragmentation path-ways of the silver complexes and the structural features of theflavonoids. The 3-O-rutinoside flavonol complexes undergo thedominant loss of the aglycon moiety followed by the loss of therhamnose residue and a few other fragmentation pathways. Thecomplexes of the flavone 7-O-rutinosides exclusively dissociateby loss of the rhamnose residue, whereas the complexes of theflavanone 7-rutinosides dissociate by other routes as well as theloss of the rhamnose residue. For each neohesperidoside/rutinoside isomeric pair, the silver complex containing theneohesperidoside typically has two or three additional diagnosticfragmentation pathways or its major fragment ions are substan-tially more intense compared to the rutinoside counterpart.Therefore, the use of silver complexation and tandem massspectrometry allows consistent differentiation of flavonoids thatdiffer only in the aglycon type (flavonol vs flavone vs flavanone)and those that differ only in the inter-saccharide linkage (rutinosevs neohesperidose). The glycosylation position also has a signifi-

Figure 8. Total ion chromatogram (TIC) and reconstructed selectedion chromatogram (SIC) of a grapefruit juice extract by LC/MS afterpostcolumn silver complexation. Figure 9. LC/MS/MS analysis of a grapefruit juice extract after

postcolumn silver complexation. 33% CAD energy was applied toeach complex ion of interest (labeled with asterisks). Losses: R )rhamnose residue (146), A ) aglycon moiety, and D ) disaccharidegroup (308).


cant impact on the CAD dissociation patterns of the silvercomplexes of flavonoids: the three flavonol-3-rutinosides haveunique fragment ions that are not shared by their 7-rutinosideand 7-neohesperidoside analogues. The silver complexation methodis also easily implemented into a versatile postcolumn com-plexation LC/MS/MS strategy for analysis of flavonoids inmixtures.

ACKNOWLEDGMENT

This work was supported by the Welch Foundation (F-1155)and the National Institutes of Health (R01-GM63512).

Received for review August 10, 2004. Accepted December22, 2004.

AC048818G


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