Application of Mass Spectrometry for Identification and Structural Studies of Flavonoid Glycosides 2000 Phytochemistry

Review

Application of mass spectrometry for identi®cation and structuralstudies of ¯avonoid glycosides

Maciej Stobiecki

Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704 PoznanÂ, Poland

Received 6 July 1999; received in revised form 23 February 2000

Abstract

Mass spectrometry is an important tool for the identi®cation and structural determination of ¯avonoid glycosides. The

advantages of mass spectrometry are high sensitivity and possibilities of hyphenation with liquid chromatographic methods forthe analysis of mixtures of compounds. Di�erent desorption ionization methods allow the analysis of underivatized glycosides. Areview of mass spectrometric techniques applied to the identi®cation and structural studies of ¯avonoid glycosides ispresented. 7 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Flavonoid glycosides; Mass spectrometry; Liquid chromatography±mass spectrometry; Desorption ionization methods

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237

2. Electron impact and chemical ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240

3. Desorption ionization techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2433.1. Field desorption (FD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243

3.2. Fast atom bombardment (FAB) and liquid secondary mass spectrometry (LSIMS). . . 2433.3. Direct chemical ionization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

4. Liquid chromatography±mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

5. Conclusion and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253

1. Introduction

Flavonoid conjugates constitute a very diverse groupof secondary metabolites in plants (Harborne, 1988a,

1988b). Most ¯avonoids in plant cells are present asglycosides. Sugar substitution on the ¯avonoid skel-eton may occur through hydroxyl groups in the caseof O-glycosides or directly to carbon atoms in ring Ain C-glycosides. The number of sugar rings substitutedon the aglycone varies from one to four. Anothergroup of polyphenols present in the plant tissue,

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mostly in wood and bark, are the proanthocyanidins(condensed tannins), polymers of ¯avan-3-ols withdi�erent degree of polymerization (Haslam, 1981; Staf-ford, 1990).

These classes of secondary metabolites play an im-portant role in interactions of plants with the environ-ment (Berhow and Vaughn, 1999). Flavonoidglycosides and free aglycones are involved in inter-actions of plants with microorganisms, both patho-genic and symbiotic (Dixon et al., 1994; Spaink, 1995;Gianinazzi-Pearson, 1996). They also act as UV pro-tectants in plant cells (Shirley, 1996), pigment sourcesfor ¯ower colouring compounds (Goto and Kondo,1991; Weiss, 1991) and they play important roles in in-teractions with insects (Nahrstedt, 1989; Harborne,1988a, 1988b). These plant metabolites also a�ect thehuman and animal health because of their signi®cancein the diet, which is ascribed to their antioxidant prop-erties (Rice-Evans et al., 1997), estrogenic action (Mik-sicek, 1993) and a wide spectrum of antimicrobial andpharmacological activities (Wollenweber, 1988; Wei-denboÈ rner et al., 1990; Dixon and Steele, 1999).

Due to the importance of ¯avonoids and their glyco-sides to and in living organisms, the identi®cation and/or structural determination of such compounds occur-ring in plant tissue or other biological systems play animportant role in many areas of science, particularly inplant science. Mass spectrometry (MS) is one of thephysico-chemical methods applied to the structural de-termination of organic compounds. The high sensi-tivity and possibilities of hyphenation withchromatographic techniques sets MS among the mostappropriate physico-chemical methods for the study ofnatural products from biological material. The charac-teristic features of MS is the use of di�erent physicalprinciples, both for sample ionization and separationof the ions, generated according to their mass (m ) tocharge (z ) ratio (m/z ). In this respect MS is di�erentfrom other spectroscopic methods and it provides con-siderable ¯exibility in the detection, quanti®cation,identi®cation and structural determination of com-pounds.

The application of MS to the analysis of ¯avonoidglycosides has increased with the new development ofso called, ``soft'' ionization techniques. Compounds ofthis class are polar, non volatile and thermally labile.Electron impact (EI), with electron energies rangingfrom 10 to 100 eV, and chemical ionization (CI) werenot suitable for MS analyses of underivatized ¯avo-noid glycosides. Both methods require the analyte tobe in the gas phase for ionization, and derivatizationof the hydroxyl groups (methylation, trimethylsilyla-tion and acetylation) was necessary. Only limited struc-tural information about the sugar or the aglyconecould be obtained for derivatized mono- and di-glyco-sides. With the introduction of desorption ionization

techniques, the analysis of ¯avonoid glycosides withoutderivatization became possible. The ®rst of these tech-niques, ®eld desorption (FD) was developed by Beckeyand Shulten (1975), but drawbacks related to the prep-aration of the sample for MS analysis, restricted its ap-plication. Another development was direct chemicalionization (DCI) (Hunt et al., 1977), in which thesample solution is deposited on the heated emitterplaced directly in the reactant gas. The ions thus gen-erated are desorbed and accelerated to the MS analy-zer. In the early 80s, two new techniques wereintroduced: fast atom bombardment (FAB) (Barber etal., 1982) and liquid secondary ions mass spectrometry(LSIMS) (Aberth and Burlingame, 1984). An extensionof these methods was the development of continuous¯ow fast atom bombardment (CF FAB/LSIMS) (Itoet al., 1985, 1986; Caprioli et al., 1986; Caprioli,1990a, 1990b). In all three ionization methods, sampledissolved in a high boiling solvents is bombarded withhigh energy atoms (argon or xenon) or cesium ions, atenergies of 8±30 keV. In parallel with these develop-ments, other techniques were introduced, that were es-pecially applicable to the combination of liquidchromatography with mass spectrometry. The mostinteresting, from the point of view of structural studiesof ¯avonoid glycosides are thermospray (TSP) (Blakleyand Vestal, 1983), and atmospheric pressure ionization(API) methods: electrospray (ESI) which is especiallysuited to the analysis of peptides and proteins (White-house et al., 1985; Fenn et al., 1989), and atmosphericpressure chemical ionization (APCI) (Horning et al.,1974). The technical problems and the low sensitivityassociated with the earlier introduced hyphenationtechniques: the moving belt (McFadden, 1980) andparticle beam (Bruins, 1985), limited seriously their ap-plicability.

An excellent review by Arpino (1989, 1990) describesthe LC/MS techniques introduced between 1980 and1989. In addition, a number of papers that deal withthe advances in instrumentation and applications ofLC/MS systems for the analysis of mixtures of second-ary metabolites from plants have been published(Maillard et al., 1993; Wolfender and Hostettmann,1993, 1997; Sato et al., 1994; Wolfender et al., 1995a,1997; Zhou and Hamburger, 1996; Vissers et al., 1997;Careri et al., 1998; Bruins, 1998; Niessen, 1998, 1999).

Flavonoid aglycones are structurally a rather diversegroup of natural products. The most important vari-ations in their structure arise from the level of oxygen-ation (hydroxyl or methoxyl groups) and the point ofattachment of ring B (¯avonoids and iso¯avonoids(Fig. 1). The sugar component may consist of hexoses,deoxyhexoses or pentoses and in some cases glucuronicacids with the added possibility of O- or C-glycosida-tion. Di�erences in the con®guration at the anomericcarbon(s) of the glycosidic units are also possible. The

M. Stobiecki / Phytochemistry 54 (2000) 237±256238

consequence of this is that single MS technique willnot provide all necessary structural information allow-ing an unambiguous assignment of the structure to anunknown compound.

Fragmentation pathway of O-glycosylated ¯avo-noids starts with the cleavage of the glycosidic bondsand elimination of the sugar moieties with chargeretention on the aglycone or sugar fragments. In C-

Fig. 1. General structure of ¯avonoids and sites of their glycosylation and methylation, position of possible glycosilation of the ¯avone aglycone

are indicated with arrows.

M. Stobiecki / Phytochemistry 54 (2000) 237±256 239

glycosides, mainly fragmentation of the sugar units isobserved. In contrast to what is observed with EI tech-niques, in normal desorption ionization mass spectrafragmentation of the aglycone is not seen. With thelater techniques (FAB, LSIMS, DCI) the informationavailable is limited to the molecular mass of the com-pound, the size of the ¯avonoid molecule and thenumber of sugar units constituting the glycoside. Inaddition, with compounds containing two or moresugars, ions arising from the cleavage of the glycosidicbonds between sugar units are frequently weak.

The structural information can be enhanced byusing tandem MS with collision induced decompo-sition (CID MS/MS) and desorption ionization tech-niques. Di�erent experimental approaches forobtaining MS/MS spectra are known (McLa�erty,1983; Jennings, 1996). They are classi®ed according tothe translational energy of ions at which the collisionsoccur. The energy of ions leaving an ion source isdependent on the type of analyzer mounted in themass spectrometer. In instruments equipped with anelectromagnetic analyzer (magnetic-B and electrostatic-E sectors) high energy collisions (1±10 keV) take place.In two sector instruments, linked scans (E/B or B/Econ®guration) or mass analyzed ion kinetic energy(MIKE) experiments (only B/E con®guration) are per-formed. High energy CID MS/MS analyses are run onthree or four sector tandem mass spectrometers withboth con®gurations of the analyzers mentioned above.On the other hand, in triplequadrupole and hybridtandem instruments (electromagnetic and quadrupoleanalyzer), low energy collisions (energy below 100 eV)are involved. Tandem mass spectrometric experimentsare also performed on instruments with ion trap analy-zers (March, 1992), and in this case, it is possible toperform tandem experiments many times (MSn) onsequential product ions. This is an interestingapproach since it permits structural information fromone single analysis.

With respect to ¯avonoid glycosides, the followinginformation can be obtained from mass spectrameasured using the techniques outlined above: (1) mol-ecular mass, (2) structure of aglycone (pattern of hy-droxylation on aglycone, point of attachment of ring Bon ring C), (3) information about acylation of sugarhydroxyl groups and possible methylation or sulpha-tion of aglycone hydroxyl(s), (4) number of sugarrings, their con®guration and in some cases placementof glycosidic bonds. MS does not provide informationabout stereochemistry of the glycosidic linkage or dis-tinguish between diastereomeric sugar units.

2. Electron impact and chemical ionization

Mass fragmentation pathways of ¯avonoid agly-

cones induced with electron bombardment ionizationare well recognized (reviewed by Mabry and Ulubelen,1980). EI mass spectrometry has been also applied tosome extent for structural investigation of O- and C-glycosylated ¯avonoids. Prior to MS analyses, thecompounds were methylated (Bouillant et al., 1975,1978, 1979, 1980, 1984; Besson et al., 1979; Sakushimaand Nishibe, 1988b), trimethylsilylated (TMS) (Wagnerand Seligmann, 1973; Schels et al., 1977, 1978) oracetylated (Sakushima et al., 1980; Sakushima andNishibe, 1988b). However, during methylation of ¯a-vanone glycosides, side reaction may take place and re-arrangement of the ¯avonoid aglycone to chalconesometime occurs (Shmid, 1972). In these cases, ad-ditional puri®cation procedures for the isolation of theproducts are necessary.

Zinsmeister and coworkers studied the mass spectralbehavior of TMS derivatives of mono-, di-, and tri-gly-cosides (Schels et al., 1977, 1978). Molecular ions wereobserved in the mass spectra, but the fragmentationpathway was dominated by fragment ions generatedafter expulsion of the TMS groups. Ions created aftercleavage of the glycosidic bond with charge retentionon the aglycone were also registered, while ions result-ing from retro Diels-Adler reactions (RDA) were notobserved. The site of the sugar substitution on theaglycone at C-7 and C-3 or C-5 could be determinedfrom the relative intensities of the fragment ions cre-ated after the glycosidic bond cleavage. Ions character-istic for sugar fragments were also observed, but theydid not bring any information about isomeric struc-tures and form of the sugar ring. EI mass spectraregistered from permethylated derivatives of ¯avonoidglycosides provided more structural information. Frag-ment ions from aglycones, including those comingfrom RDA reactions were observed, permitting eluci-dation of the structure of the ¯avonoid skeleton. Simi-larly, di�erentiation of hexoses, pentoses andglucoronic acid was also possible (Bouillant et al.,1975). The di�erences in the intensities of molecularand fragment ions in the mass spectra of some ¯avo-noid glycosides were used to determine the location ofthe sugar ring (at C-7, C-3, or C-5). On the samebasis, di�erentiation of sugar substitution at C-6 or C-8 of the ¯avonoid moiety was also possible in the caseof C-glycosides (see Fig. 2). This was concluded fromthe di�erences in the relative intensities of M+� andfragment created after cleavage of CH3O

� radical [M-31]+ in the spectra of permethylated (PM) isomeric C-glycosides of both luteolin and apigenin. Di�erences inthe MS spectra of C-6 and C-8 disubstituted glycosidesresulted from sequential elimination of sugar frag-ments, and in the case of two di�erent sugars (hexoseand deoxyhexose or hexose and pentose) it was poss-ible to ascribe the positions at which they were substi-tuted. On the contrary, only low intensities of


molecular ions were registered in the mass spectra ofacetylated ¯avonoid glycosides. The fragmentationpathway was dominated by sequential elimination ofketene molecules or acetyl radicals (Sakushima et al.,1980).

The application of chemical ionization for studies of¯avonoid glycosides did not give satisfactory results.

When methane or ammonia were used as reagent gas,the protonated molecular ions were not observed inmass spectra of unmodi®ed compounds. However,with the use of amines, it was possible to detect proto-nated molecules of several mono-O-glycosides (Mol-lowa et al., 1987). Although negative CI has also beenused, only poor results were obtained, due to low

Fig. 2. Electron impact mass spectra of permethylated ¯avonoid C-glycosides. PM Ð permethylated compound (reprinted with permission from

Bouillant et al., 1975).


Fig. 3. Positive ions FAB mass spectra of ¯avonoid triglycosides: a Ð kaempferol 3-O-(60-O-rhamnosyl)-galactoside 7-O-glucoside, b Ð kaemp-

ferol 3-O-(60-O-rhamnosyl)-galactoside 7-O-rhamnoside (robinin). Ym+n : fragment ion created after cleavage of glycosidic bond between sugars;

superscript describes the place of substitution on the aglycone; subscript describes the distance of the glycosidic bond from the aglycone; Y0+: an

aglycon ion; (Ymn /Y

mn )

+: fragment ion created after cleavages of two sugars substituted in two di�erent positions on an aglycone. G: ions origi-

nated from protonated clusters of glycerol used as a matrix (reprinted with permission from Sakushima and Nishibe, 1988a).


intensities or lack of [M + H]+ ions in the registeredmass spectra (Itokawa et al., 1982).

3. Desorption ionization techniques

When applied to various unmodi®ed ¯avonoid gly-cosides, desorption ionization techniques enabled thegeneration of mass spectra for molecules containing upto four sugar units, both in the positive and the nega-tive ions mode. Intense ions are usually registered forprotonated and/or cationized molecules [M + H]+,[M + Na]+, [M + K]+ (positive ions mode) ordeprotonated molecules [M ÿ H]ÿ� (negative ionsmode). The fragmentation pathway follows the clea-vage of glycosidic bonds in O-glycosides, consecutiveexpulsion of sugars is observed, and ions originatingfrom the aglycones are also registered (Fig. 3). Itshould be noted however, that in the desorption spec-tra (FAB and LSIMS) of organic compounds usuallyions of protonated clusters of matrix molecules arefound, for example glycerol (G ions in Fig. 3). In themass spectra of kaempferol triglycosides there wereobserved ions of the protonated cluster of glycerolmolecules, used as matrix. As to C-glycosides, the frag-mentation takes place in di�erent ways. Mainly thecleavage of the sugar moiety is observed, sometimeswith an additional fragmentation of the aglycone,according to the retro RDA. The relative intensities ofthe sample ions in the mass spectra registered with de-sorption ionization methods depend on the ionizationtechnique used, the structure of the compound studied,and the purity of the sample.

3.1. Field desorption (FD)

This was the ®rst technique applied for the analysisof intact ¯avonoid glycosides (Geiger and Schwinger,1980; Domon and Hostettmann, 1985; Stein et al.,1985; Barbera et al., 1986). In all cases, the highestintensities were observed for ions of cationized mol-ecules [M + Na]+ and [M + K]+, while ions of pro-tonated molecules were of lower intensity. Fragmentions of di�erent intensities, created after the consecu-tive loss of sugar moieties were also observed. It waspossible to identify ions originating from the aglyconemoiety, so the aglycone ions [A]+, [A + H]+ and [A+ Na]+ showed substantial intensities, in some spec-tra also [A]2+ ions. During studies on acylated glyco-sides from Bryum cappilare, ®eld desorption massspectrometry was also used to con®rm the substitutionof the sugar moiety with a malonyl group (Stein et al.,1985).

3.2. Fast atom bombardment (FAB) and liquidsecondary mass spectrometry (LSIMS)

There are numerous examples on the application ofFAB and LSIMS for the analysis of ¯avonoid glyco-sides, both in positive and negative ion modes (Saitoet al., 1983, 1985; Bridle et al., 1983; Domon et al.,1985; Crow et al., 1986; Sakushima et al., 1988; Sto-biecki et al., 1988; Sakushima and Nishibe, 1988a; Bec-chi and Fraisse, 1989; Li et al., 1991, 1992a, 1992b;Wolfender et al., 1992; Li and Claeys, 1994; Claeys etal., 1996; Sumner et al., 1996). These ionization tech-niques were also used for structural studies of con-densed tannins (Gujer et al., 1986; Self et al., 1986;Ohnishi-Kameyama et al., 1997). In the mass spectra,ions of protonated [M + H]+ or deprotonated [M ÿH]ÿ� molecules of both classes of compounds wereobserved, as well as fragment ions. In the mass spectraof ¯avonoid glycosides, fragments were created aftercleavage of bonds between sugars or sugar and agly-cone, and in the later case between proanthocyanidinunits.

The mass spectra recorded with both FAB andLSIMS techniques are often burdened with ions orig-inating from the matrix used. Additionally, in somecases low signal-to-noise ratio (S/N) for protonatedmolecules and fragment ions is also observed. Thematrices most often used (high-boiling solvents) areglycerol, nitrobenzyl alcohol (NBA), dithiothreitol/ditioerythritol, 5:1 w/w (magic bullet, DTT/DTE) andthioglycerol. The most suitable matrices are the ®rstthree mentioned solvents, because of long lasting sig-nals, originating from the compounds analyzed, in theion source of the mass spectrometer. This is especiallyimportant when CID MS/MS experiments are per-formed. A proper choice of the matrix a�ects also theyield of analyte ions sputtered from the solution (DePauw, 1986; van der Peyl et al., 1985). Possibleincrease of the S/N ratio resulting for compound ions,improves signi®cantly the quality of FAB spectra. Pro-blems arising from the observed high intensities ofmatrix ions can be overcome with the use of continu-ous ¯ow fast atom bombardment (CF FAB/LSIMS).This was clearly demonstrated by comparative analysesof rutin with LSIMS and CF LSIMS desorption tech-niques applied for registration of the mass spectra of¯avonoid glycosides (Sumner et al., 1996), see Fig. 4.

Another strategy aiming to improve the S/N ratio inFAB spectra is methylation of the ¯avonoid glycosidesprior to the MS analysis (Stobiecki et al., 1988). Thisapproach provides also an additional informationabout the substitution pattern of sugars on the agly-cone. The masses of partially methylated aglycone ionscreated after cleavage of the sugar moieties couldeasily be identi®ed, the resulting di�erence in the massbetween unmethylated and methylated aglycone


enables the determination of the pattern of sugars sub-stitution on the aglycone. The mass spectra of unmodi-®ed and permethylated quercetin 3-O-galactoside-7-O-glucoside are demonstrated on Fig. 5, the intense frag-ment ions (Yn

+) were registered in the mass spectrumof permethylated glycoside.

The importance of the proper choice of matrix isalso important because signals from protonated clus-ters of matrix molecules can overlap with ions of theprotonated analyte molecules or with fragment ionsoriginating from the ¯avonoid glycosides. In this casethe use of a few di�erent matrices is recommended.High concentration of sodium cations in the samplesmay also decrease the observed intensities of ions cre-ated from the molecules analyzed (Li et al., 1992b;Claeys et al., 1996). Thus, prior to MS analyses,samples of ¯avonoid glycosides should be puri®ed onreversed phase C-18 cartridges with diluted acid as asolvent. The importance of such approach is illustratedin Fig. 6. Other impurities present in the samples, suchas phthalates or polyethylene glycols, originating fromthe solvent for the isolation of the compound, alsonegatively in¯uence the intensities of the analyte ions.

Hexoses, deoxyhexoses and pentoses could be di�er-entiated in normal desorption (FAB or LSIMS) massspectra of unmodi®ed ¯avonoid O-glycosides, on thebasis of m/z values of fragment ions created after clea-vage of the glycosidic bonds. However, such fragmentions have often very low intensities and their identi®-cation between matrix ions is usually di�cult. In suchsituations application of collision-induced dissociationtandem MS techniques (CID MS/MS) is very useful.For example, Crow et al. (1986) registered high energyCID MS/MS spectra of rutin and naringin on a threesector instrument (electric/magnetic/electric Ð E/B/E),where a collision cell was placed between the magneticand the second electric sector. The comparison of nor-mal and CID MS/MS spectra revealed substantialdi�erences in the intensities of fragment ions createdafter the elimination of the sugar moieties. This di�er-ence was especially pronounced in the mass spectra ofnaringin, in which the glycosidic bond between rham-nose and glucose is situated at C-1 and C-2. Only [M+ H]+ ion at m/z 581 was observed in the normalFAB spectrum, while the fragments created after clea-vage of the glycosidic bonds: Y0

+ at m/z 273 and Y1+

at m/z 435, were registered during FAB CID MS/MSexperiments. Similarly, structurally signi®cant di�er-ences have been also found in the intensities of frag-ment ions created after degradation of the sugar ringin hesperidin (1±2 glycosidic bond) and neohesperidin(1±6 glycosidic bond) with the use of CID negative ionMS/MS technique (Fig. 7).

Other MS/MS techniques have also been applied forstructural studies of ¯avonoid glycosides. Becchi andFraisse (1989) demonstrated the usefulness of the col-

Fig. 4. CF-FAB mass spectra of rutin [quercetin 3-O-(60-O-rhamno-

syl)-galactoside]: a Ð positive ion LSIMS mass spectrum; b Ð posi-

tive ion CF LSIMS mass spectrum; c Ð negative ion CF LSIMS

mass spectrum; Yn+ and Zn

+ fragment ions created after cleavages of

glycosideic bonds (reprinted with permission from Sumner et al.,

1996).


Fig. 5. FAB mass spectra of quercetin 3-O-galactoside 7-O-glucoside: a Ð unmodi®ed; b Ð permethylated; Ym+n Ð fragment ions created after

cleavages of glycosidic bonds (reprinted with permission from Stobiecki et al., 1988).


lision-induced dissociation mass analyzed ion kineticenergy (CID/MIKE) technique for the analysis of C-6and/or C-8 mono- and di-glucosides isolated fromplant tissues. In this case, the mass spectra in the nega-

tive ion mode were recorded on a reversed geometry(B/E) mass spectrometer. The same technique usedStobiecki et al. (1988) to analyse the permethylatedquercetin 3-O-xylosyl-galactoside. On the other hand,

Fig. 6. FAB mass spectra of diglucoside kaempferol 3-O-(60-O-rhamnosyl) glucoside: a Ð no desalting; b Ð desalted with deionized water; c Ð

desalted with 0.02M HCl; Yn+: fragment ions created after cleavages of glycosidic bonds (reprinted with permission from Li et al., 1992b).


Claeys and co-workers (Li et al., 1991, 1992a, 1994;Claeys et al., 1996) applied a hybrid instrument withquadrupole as second analyzer (EBqQ) for characteriz-ation of O- and C-substituted ¯avonoid glycosides. Inthis case, the desorption ionization CID spectra wereusually registered in a positive ion mode due to thebetter sensitivity observed. High and low energy exper-iments were performed. In the ®rst case, collisionswere induced with helium in the ®rst ®eld-free regionwith an energy of 8 keV and 50% beam attenuation,and product ions linked scan (B/E = const) mass spec-tra were registered. This enabled the determination ofsugar substitution pattern of isomeric C-6 and C-8 glu-cosides, isoorientin and orientin respectively, on the

basis of the abundance di�erences observed for theproduct ions in the CID MS/MS mass spectra (Li etal., 1992a) (Fig. 8). For recording low energy CIDmass spectra precursor ions were selected at the resol-ution 1000 in a double focusing mass spectrometer;collisions were performed at an energy of 50 eVbetween the electromagnetic and the quadrupole analy-zers. The low energy CID MS/MS spectra wererecorded not only for protonated molecules of glyco-sides, but also for fragment ions, corresponding toaglycone ions created after cleavage of the sugar moi-eties. FAB CID MS/MS spectra with low energy col-lisions of protonated molecular ion of kaempferolrutinoside [M + H]+ at m/z 595 and fragment ion of

Fig. 7. CID MS/MS mass spectra: a Ð hesperedin Ð hesperetin 7-O-(60-O-rhamnosyl) glucoside; b Ð neohesperidin Ð hesperetin 7-O-(20-O-

rhamnosyl) glucoside (reprinted with permission from Crow et al., 1986).


apigenin at m/z 271 present in the mass spectrum ofapigenin 7-O-neohesperidoside are demonstrated inFig. 9.

Stobiecki and coworkers presented a methodologicalapproach where di�erent mass spectrometric tech-niques (LSIMS normal and CID linked scan spectra,EI GC/MS of chemically modi®ed compounds) wereapplied for structural studies of ¯avonoid glycosidesisolated from Lupinus luteus (FranÂ ski et al., 1999). Thegeneral scheme describing application of di�erent massspectrometric techniques in combination with the useof simple chemical modi®cations of compounds stu-died is shown in Fig. 10. In this approach, an unam-biguous identi®cation of aglycones and sugars, as wellas the con®rmation of the positions of glycosidicbonds, is achieved after GC/MS analyses of chemicallymodi®ed compounds (permethylated, methanolyzed in1 N HCl, and again methylated or acetylated). A simi-lar approach for the determination of sugar substi-tution on ¯avonoid skeleton was proposed bySakushima and Nishibe (1990). In this case, however,

monotrimethylsilylated ¯avonoid methyl ethers,obtained from model compounds were analysed. Otherstrategies of chemical modi®cation after hydrolysiswere also applied. They involved reduction of sugarsto alditols followed by acetylation, or trimethylsilyla-tion of the obtained products. Such protocols wereused for the elucidation of sugar moieties in anthocya-nin and ¯avonol glycosides with GC/MS methods, re-spectively (Stobiecki et al., 1988; GlaÈ ssgen et al.,1992c).

Fig. 8. High energy CID FAB mass spectra from [M + H]+ ions at

m/z 449 of C-glycosides: a Ð iso-orientin (luteolin 8-C-glucoside); b

Ð orientin (luteolin 6-C-glucoside); (both isomers can be distin-

guished on the basis of abundances of the ions at m/z 395 and m/z

353 (reprinted by permission of Oxford University Press from Claeys

et al., 1996).

Fig. 9. Low energy FAB CID MS/MS mass spectra: a Ð from [M

+ H]+ ions (595 m/z ) of kaempferol 3-O-rutinoside; b Ð from Y0+

fragment ions (271 m/z ) of apigenin neohesperoside; c Ð fragmenta-

tion pathway of apigenin; Yn+: fragment ions created after cleavages

of glycosidic bonds (reprinted by permission of Oxford University

Press from Clayes et al., 1996).


3.3. Direct chemical ionization

The DCI mass spectra of ¯avonoid glycosides inpositive and negative ion mode were presented byDomon and Hostettmann (1985). The ionizationmethod applied is applicable for ¯avonoid glycosidescontaining up to three sugar units, but the presence ofglucuronic acid is excluded. When ammonia was usedas reaction gas, protonated molecule ions [M + H]+

and ammonium adduct ions [M + NH4]+ were

observed in the mass spectra, and the presence of bothtypes of ions, mentioned above was used to determinethe molecular weight of the compounds studied. Inthis kind of mass spectra, fragments originating fromthe sugar moiety of the molecule have also beenobserved. Better results were usually obtained in thespectra registered in the negative ion mode (Domonand Hostettmann, 1985). Comparison of positive andnegative ions DCI mass spectra of robinin showedsome di�erences in the presence and/or relative intensi-ties of the molecular and the fragment ions. The [M +H]+ ion of the intact robinin molecule were not regis-tered. Only in the negative ion mass spectrum wasobserved a peak of [M ÿ H]ÿ� ions, and the main frag-mentation pattern was related to the cleavage of theglycosidic bonds (Fig. 11). Flavonoid O- and C-glyco-sides were also analysed by the negative ion DCI spec-tra, using methane as a reagent gas (Sakushima et al.,

1989). The signals from Mÿ� ions had fairly high inten-sities for monoglycosides, but decreased for di- and tri-glycosides. Fragment ions showing charge retention onsugar moieties were also identi®ed with this method.

Direct introduction electrospray mass spectrometryseems to be a valuable technique for the structuralanalysis of ¯avonoid glycosides. The mass spectra ofpure compounds introduced in neutral solvents to theelectrospray source are usually dominated by sodiatedmolecule ions [M + Na]+, while protonated moleculeions [M + H]+ often are not observed or have verylow intensities. The intensity of cationized moleculeions can be decreased by acidi®cation of the solventused (Zhou and Hamburger, 1996; Stobiecki et al.,1999). The achievable sensitivity of ESI techniquesmay be even two orders of magnitude higher thanthose of FAB or LSIMS. Another advantage of elec-trospray ionization is better S/N ratio, due to lack ofstrong ions originating from the matrix in the spectralrange above 200 amu. When using the ESI source, it isusually possible to register fragment ions created afterconsecutive cleavages of glycosidic bonds (Yn

+ ions inFig. 12). The intensities of fragment ions can be ad-ditionally increased by a collision induced dissociationin the source, achievable with an increase of the poten-tial di�erence between nozzle and skimmer in the ESIsource (Niessen, 1998, 1999).

In normal mass spectra of ¯avonoid glycosides regis-tered with di�erent desorption ionization techniques(LSIMS or FAB, DCI and FD) or direct introductionelectrospray, the observed fragmentation pathways arevery similar. Mainly glycosidic bonds are cleaved.However, in many cases the respective fragment ionsare missing due to impurities present in the samples orto physico-chemical properties of compounds understudy. In such a situation, CID MS/MS techniques arehelpful. Additionally, the application of high and/orlow energy collision experiments may give comple-menting results. The later technique is especially usefulwhen the aglycone fragmentation has to be induced(Claeys, 1996), see Fig. 9.

4. Liquid chromatography±mass spectrometry

Technical developments in coupling mass spec-trometers with liquid chromatographs, achieved duringlast decades, enabled the separation and identi®cationof many classes of polar compounds, among others ¯a-vonoid glycosides, often present as complex mixturesin plant extracts. During the ®rst attempts, only mix-tures of free ¯avonoid aglycones, isolated from biologi-cal material, were e�ciently analyzed on moving beltand particle beam interfaces with EI and/or CI ioniz-ation (Games and Martinez, 1989; Chavez et al.,1998). Application of these systems for analysis of ¯a-

Fig. 10. Proposed strategy for the application of mass spectrometric

techniques for structural studies of ¯avonoid glycosides.


vonoid glycosides did not give promising results,because of the polarity and thermolability of suchcompounds (Weinberg et al., 1992; Careri et al., 1998).

For a few years LC/MS systems have been appliedfor detection and identi®cation of ¯avonoid glycosidesin plants extracts or various biological ¯uids. Introduc-tion of thermospray ionization interface (TSP) to LC/MS systems, has enabled the analysis of mixtures ofpolar ¯avonoid glycosides (Wolfender et al., 1995a,

1995b). However, the application of this technique hassome limitations related to the thermal stability of thecompounds studied. This is connected to the high tem-perature in the TSP ion source, which is necessary fore�cient ionization of the molecules to be analysed.Additionally, optimization of the TSP ionizationsource parameters separately for each class of com-pounds in the mixture is necessary, as it stronglydepends on the LC mobile phase composition and

Fig. 11. DCI mass spectra of robinin [kaempferol 3-O-(60-O-rhamnosyl)-galactoside 7-O-rhamnoside]: a Ð positive ions, b Ð negative ions (rep-

rinted with permission from Domon and Hostettmann, 1985), Ym+(ÿ�)n : fragment ion created after cleavage of glycosidic bond between sugars;

superscript describes the place of substitution on the aglycone; subscript describes the distance of the glycosidic bond from the aglycone, Y0+(ÿ�)

an aglycon ion, (Ymn /Y

mn )ÿ�: fragment ion created after cleavages of two sugars substituted in two di�erent positions on an aglycone.


¯ow rate. Attention has also to be paid to the follow-ing parameters: vaporizer and source block tempera-tures, ammonium acetate concentration in the mobilephase introduced to the ion source (possibly post-col-umn addition to the mobile phase with an additionalpump), and repeller potential. The mass spectra regis-tered in the positive ion mode after TSP ionization aresimilar to those obtained with direct chemical ioniz-ation when ammonia is used as reagent gas. Such TSPspectra registered in positive or negative ion mode,provide information about the molecular masses of the

¯avonoid glycosides and the sizes of aglycone andsugars. Intensities of protonated molecule ions [M +H]+ in the mass spectra are dependent on the numberof sugars linked to the aglycone moiety. There is asubstantial decrease in [M + H]+ ions intensity withincreasing number of attached sugar rings, a maximumof three sugar units may be present in the studied mol-ecule. Cationized molecules with NH4

+, Na+, or K+

cations, are often registered, and adducts with com-ponents of the mobile phase are also observed. Ad-ditionally TSP mass spectra usually give fragment ionsgiving sugar sequence information according to thesize of the sugar rings (Fig. 13). LC TSP/MS has beenused for the analysis of crude plant extracts fromdi�erent sources. Hostettmann and coworkers appliedthe technique for the identi®cation of secondarymetabolites, among others ¯avonoid glycosides, inextracts from dried medicinal plants (Bashir et al.,1991; Maillard et al., 1993; Wolfender et al., 1993,1995a, 1995b, 1997; Wolfender and Hostettmann,1993, 1997). Pietta et al. (1994) carried out TSP/MSanalyses of pure ¯avonoid glycosides obtained fromGinko biloba and Calendula o�cinalis, introduceddirectly or through liquid chromatograph to the ionsource in the positive and the negative ion mode. LCTSP/MS was also successfully applied for detectionand identi®cation of a wide range of phenolic com-pounds, among others O- and C-¯avonoid mono- anddiglycosides in lemon peel (Baldi et al., 1995). Anotherexample was LC TSP/MS analysis of the extract from

Fig. 12. Direct ESI mass spectrum of rutin [quercetin 3-O-(60-O-rhamnosyl) glucoside]. Yn+: fragment ions created after cleavages of glycosidic

bonds (reprinted with permission from Stobiecki et al., 1999).

Fig. 13. TSP mass spectrum of triglycoside: kaempferol 3-O-[(20-O-

apiosyl) 60-O-rhamnosyl]-galactoside. Y0+: fragment ions created

after cleavages of glycosidic bonds (reprinted with permission from

Wolfender et al., 1993).


medicinal plant Hypericum perforatum, where a seriesof ¯avonoid glycosides was identi®ed (Brolis et al.,1998). Other group used this LC/MS method foridenti®cation of monoglycosides in extracts fromArnica montana and Arnica chamissonis (SchroÈ der andMerford, 1991). Green tea polyphenols and their gly-cosides were also studied with LC TSP MS/MS; a few¯avonoid glycosides and catechins were identi®ed (Linet al., 1993; Poon, 1998).

Liquid chromatography with CF-FAB or CF-LSIMS interfaces has also been applied for the analy-sis of plant secondary metabolites. In this method onlya very small volume of mobile phase eluted from thecolumn enters the ion source of the mass spectrometerwith maximum 10 ml/min delivered to the probe tip.During the analysis glycerol, used as matrix, wasadded to the mobile phase or delivered post-column tothe eluate (Ito et al., 1985; Caprioli, 1990). This tech-nique was used to analyse pro®les of acylated iso¯avo-noid glycosides present in alfalfa (Medicago sativa )and chick pea (Cicer arientium ). MS analyses wereperformed following puri®cation of the compounds ofinterest on a polyamide column (Sumner et al., 1996).

Since electrospray ionization (ESI) and atmosphericpressure chemical ionization (APCI) were introducedin the ®eld of combined liquid chromatography±massspectrometry, the number of papers describing theirusage has been increasing each year. Electrospray hasbecome the most popular ionization method used. At-mospheric pressure ionization systems may be coupledto di�erent mass spectrometric analyzers (quadroupole,ion trap, electromagnetic, time of ¯ight and Fourier-transform ion cyclotron resonance), and thus, di�erentdesigns of API interfaces for all kind of instrumentsare available (Niessen, 1998, 1999). Mobile phase ¯owin API interfaces may di�er from nanoliters (so callednanoelectrospray) to 2 ml per minute. The temperaturecontrol of the APCI desolvation process is far lesscritical than in TSP-MS. In this way, a wide range ofcompounds may be analysed under the same con-ditions maintained at the APCI interface. For mostmass spectrometers with electrospray ionization com-bined with LC, lower ¯ow rates, up to maximum 100ml/min are used. In many cases, splitting of the eluatefrom the LC column is necessary in order to decreasethe volume of solution entering the API source. Such a¯ow rate enables an e�cient desolvation process. Split-ting of the column eluate provides also an opportunityto use double detection systems (UV and MS) of com-pounds present in the sample (Wolfender et al., 1995a,1997, 1998). The mechanisms of electrospray and at-mospheric pressure chemical ionization di�er (Zhouand Hamburger, 1996). Both ionization processes arebased on di�erent physico-chemical e�ects. In ESI,ions are preformed in solution and this is essential forthe next steps of ion formation in the gas phase. On

the other hand, APCI relies on the gas phase chemistrywhere molecules have to be vaporized into the gasphase prior to ionization through charge or protontransfer. Additional feature of an electrospray ioniz-ation is in-source collision induced dissociation (CID)of protonated molecules obtained with potential di�er-ence (30±50 V) between nozzle and skimmer (Niessen,1998). This enhancement of fragmentation may be alsoapplied in structural elucidation of ¯avonoid glyco-sides. LC-ESI/MS has been applied for the identi®-cation of ¯avonoid glycosides isolated from di�erentbiological sources, for example in foods, (reviewed byCareri et al., 1998). GlaÈ ssgen et al. (1992a, 1992b) usedLC-ESI/MS and LC-ESI/MS/MS for the identi®cationof anthocyanin glycosides in plant tissue and cell cul-tures of Daucus carota. More than 10 compounds wereidenti®ed, among them mono-, di- and tri-glycosides.In some cases, terminal sugars were found to be acy-lated with sinapic, ferulic, or ca�eic acid (GlaÈ ssgen etal., 1992a, 1992b). The same LC-ESI/MS system wasused for the detection and determination of the com-position of ¯avonoids and their glycosides inOenothera (Neumann and Schwemmle, 1993). The LC-ESI/MS system was also applied for a selective screen-ing of 6 '-O-malonylated or acetylated glucoconjugatespresent in plant tissues (fruits, roots, leaves) of specieswhich are included in human diet (Withopf et al.,1997), or in foods prepared from soy (Barnes et al.,1994) and tomatoes (Mauri et al., 1999). Qualitativeand quantitative analyses of free ¯avonoids and theirglycosides in vegetables and beverages were performedwith mixed, photo-diode array and mass spectrometricdetection (Justesen et al., 1998; Ryan et al., 1999). Thesame analytical strategy was applied for the identi®-cation of ¯avonol aglycones and glycosides in berries(HaÈ kkinen and Auriola, 1998). Finally, the ®rstattempts for a parallel application of mass spec-trometry, nuclear magnetic resonance and UV detec-tion for the identi®cation of ¯avonoid glycosides incrude plant extract have been reported by Hostett-mann and co-workers (Hostettmann and Wolfender,1997; Wolfender et al., 1998).

5. Conclusion and perspectives

During the last decade mass spectrometry hasrapidly evolved to an instrumental method which canbe used in chemical and biological laboratories. Manydedicated instruments are now available on the mar-ket, most often they are combined with liquid chroma-tography. Automation of both systems is readilyachieved. The LC/MS systems can be applied forscreening of ¯avonoid glycosides in plant tissue or bio-logical ¯uids of di�erent origin. The method is es-


pecially valuable when acylation of ¯avonoid glyco-sides is investigated.

Mass spectrometric techniques in combination withchemical derivatization of compounds may be appliedfor structural studies of ¯avonoid glycosides down tosubmilligram amounts.

New developments in the ®eld of mass spectrometryfacilitate the application of such methods in plant andmedicinal sciences, but in some cases, the skill of anexperienced mass spectrometrist, especially in CIDMS/MS, is necessary.

Acknowledgements

The author is grateful to Dr P. Wojtaszek and thereferee for their valuable remarks and English correc-tions of the manuscript.

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Application of Mass Spectrometry for Identification and Structural Studies of Flavonoid Glycosides 2000 Phytochemistry