studies on the homolytic and heterolytic cleavage of kaempferol and kaempferide glycosides using...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2010; 24: 169–172

ublished online in Wiley InterScience (www.interscience.wiley.com) DO
P
RCM

Letter to the Editor

Dear Editor,

Studies on the homolytic and heterolytic cleavage of

kaempferol and kaempferide glycosides using electrospray

ionization tandem mass spectrometry

In the analysis of flavonoid glycosides, liquid chromatog-

raphy coupled with mass spectrometry (LC/MS) has proved

to be a powerful technique for rapid identification and

characterization of flavonoid glycosides in complex mix-

tures.1,2 As summarized in several recent reviews,3–5 mass

spectrometry, especially electrospray ionization tandem

mass spectrometry (ESI-MSn), is recognized as an important

tool in the structural elucidation of flavonoid glycosides.6–17

A common fragmentation process of deprotonated flavonoid

glycosides ([M–H]�) is the loss of a glycan residue to generate

the aglycone fragment ion [Y0]� (heterolytic cleavage). A

systematic study of the collision-induced dissociation (CID) of

flavonoid mono-O-glycosides has revealed that the deproto-

nated flavonoid glycosides underwent not only the conven-

tional heterolytic cleavage, but also homolytic cleavage,

yielding the aglycone fragment ion [Y0]� and the radical

aglycone ion [Y0–H]�., respectively.18 Thereafter, there was

growing interest in studies of the radical aglycone ions in CID

experiments of the deprotonated flavonoid glycosides.19–23

Compared with flavonol 7-O-glycosides, flavonol 3-O-glyco-

sides have been reported to yield homolytic cleavage

fragments in large amounts. Even the relative abundance

ratio of [Y0–H]�.

to [Y0]� has been proposed as a diagnostic

criterion to determine the glycosylation position of flavonol

glycosides.24–26 Herein, the fragmentations of a series of

kaempferol and kaempferide glycosides were studied using

negative ion tandem mass spectrometry. Our results suggested

that the length of the saccharide chains at the 3-O position of

the flavonol glycosides also played an important role in the

homolytic and heterolytic cleavage of deprotonated flavonol

glycosides and one should take precautions in the structural

elucidation of flavonoid glycosides using tandem mass

spectrometry.

All mass spectrometry experiments in this study were

performed on a Finnigan LCQ ion trap mass spectrometer

equipped with an ESI source in negative mode. The

instrumental parameters were optimized and set as follows:

nitrogen was used as desolvation gas at a flow of 40 L/h, the

metal capillary temperature was set at 2108C, the spray

voltage was set at �4.2 kV, the capillary voltage was �35 V

and the tube lens offset was �15 V. For CID experiments,

helium was used as the collision gas and the isolation width

of precursor ions was set as 1 Da. The flavonol glycosides

(Scheme 1) including kaempferol 7-O-a-L-rhamnoside,

kaempferol 3-O-b-D-glucoside, kaempferol 3-O-rutinoside,

kaempferol 3-O-b-D-glucosyl-(1!6)-a-L-rhamnosyl-(1!2)-

a-L-rhamnoside, kaempferide 7-O-a-L-rhamnoside, kaemp-

feride 3-O-b-D-glucoside and kaempferide 3-O-rutinoside

were isolated from Actinidia kolomikta and their structures

were identified by spectroscopic methods.27 The flavonols

were dissolved in methanol and introduced into the ESI

source with a syringe pump at 5mL/min.

The nomenclature rule for flavonoid aglycones developed

by Mabry and Markham28 was used for definition of the

various A- and B-ring fragments. The numbering scheme for

substitution and the ring bonds are labeled with large and

small font in Scheme 2, respectively. The i,jA� labels

designate primary product ions containing an A-ring.

Although kaempferol 3-O-glycosides and kaempferol 7-O-

glycosides produced different UV spectra in liquid chroma-

tography, we aimed to differentiate them by tandem mass

spectrometry. Figure 1 shows the MSn spectra of kaempferol

3-O-glucoside and kaempferol 7-O-rhamnoside. It was

clearly observed from the MS/MS spectra (Figs. 1(a)

and 1(c)) that homolytic and heterolytic cleavage occurred

to different extents depending on the glycosylation site. For

kaempferol 3-O-glucoside, homolytic cleavage was predo-

minant, resulting in a larger amount of the radical aglycone

ion [Y0–H]�.

(m/z 284). However, for the deprotonated

kaempferol 7-O-rhamnoside, the conventional heterolytic

cleavage occurred to a larger extent, yielding abundant

aglycone fragment ion [Y0]� (m/z 285).

The results were consistent with previous studies,18,23–26

which proposed that flavonol 3-O-glycosides favored the

homolytic cleavage and assumed the glycosylation site could

be deduced from the relative abundance ratio of [Y0]� to

[Y0–H]�.

ions. The [Y0–H]�.

ion (m/z 284) from kaempferol

3-O-glucoside and the [Y0]� ion (m/z 285) from kaempferol

7-O-rhamnoside were subjected to further CID experiments.

Different MS3 spectra were obtained: the [Y0–H]�.

ion

(m/z 284) mainly lost COH.

to generate an ion at m/z 255;

whereas, the [Y0]� ion (m/z 285) lost CO to yield an ion at

m/z 257 and the ion at m/z 151 dominated the product ion

spectrum which was attributed to the 1,3A� ion.

Besides kaempferol 3-O-glucoside, kaempferol 3-O-rutino-

side and kaempferol 3-O-b-D-glucosyl-(1!6)-a-L-rhamno-

syl-(1!2)-b-D-glucoside were also analyzed by tandem

mass spectrometry (Figs. 2(a) and 2(b)). In contrast to

kaempferol 3-O-glucoside, these two kaempferol 3-O-

glycosides underwent a predominant heterolytic cleavage

to generated abundant aglycone fragment ions [Y0]� (m/z

285). This fragmentation pathway was similar to kaempferol

7-O-glycoside (Fig. 1(c)). In this instance, it is hard to clearly

figure out the glycosylation site for an unknown kaempferol

glycoside solely from the analysis of its MS/MS data. Further

fragmentation of the [Y0]� ion (m/z 285) generated from

kaempferol 3-O-triglycoside (Fig. 2(c)) caused a loss of CO

and H2O to generate ions at m/z 257 and 267, respectively.

Moreover, the ion at m/z 163 which was ascribed to a 0,2A�

ion appeared in the spectrum, which was quite different from

that of kaempferol 7-O-rhamnoside. The fragment ion [Y0]�

(m/z 285) generated from kaempferol 3-O-di-glycosides

(kaempferol 3-O-rutinoside) exhibited a similar fragmenta-

I: 10.1002/rcm.4368

Copyright # 2009 John Wiley & Sons, Ltd.

Scheme 1. Structure of flavonoid glycosides.

Scheme 2. Structure of kaempferol, nomenclature and prin-

cipal fragmentations.

170 Letter to the Editor

tion pattern to that generated from tri-glycosides (data not

shown). The glycosylation site could be readily determined

using tandem mass spectrometry.

The formation of the fragment ion 1,3A� involved a retro-

Diels-Alder (RDA) fragmentation. In the transition state

depicted in Scheme 3(a), the electron transfers from bonds 1

and 3 to bonds 0 and 4. However, the 0,2A� ion is formed via

another RDA fragmentation that involves scission at bonds 0

and 2, as explained in Scheme 3(b). Deprotonated kaempferol

3-O-glycosides have a resonated structure which forms a

double bond at bond 4, and the resonated structure is

capable of carrying out a RDA reaction to produce the RDA

fragment ion 0,2A�.

Similar results were obtained from the analysis of kaemp-

feride glycosides. The MSn spectra of kaempferide 3-O-

Figure 1. CID spectra of kaempferol mono

kaempferol 3-O-glucoside, (b) MS3 spectrum

[Y0–H]�. ion (m/z 284) as precursor, (c) MS/MS

(d) MS3 spectrum of kaempferol 7-O-rhamno

precursor. AI denotes the absolute intensity of


glucoside and kaempferide 7-O-rhamnoside are shown in

Supplementary Fig. S1 (see Supporting Information). The

fragmentation pathways of kaempferide mono-O-glycosides

were similar to those of kaempferol mono-O-glycosides.

Kaempferide 3-O-glycosides mainly yielded a radical [Y0–

H]�.

ion at m/z 298 through homolytic cleavage, whereas

kaempferide 7-O-glycosides underwent a predominant

heterolytic cleavage to generate the [Y0]� ion at m/z 299.

Upon CID, the [Y0–H]�.

ion (m/z 298) from kaempferide 3-O-

glucoside and the [Y0]� ion (m/z 299) from kaempferide 7-O-

rhamnoside shared a specific loss of CH3.

to produce ions at

m/z 283 and 284, respectively. For further fragmentation

(MS4), the [Y0–H–CH3]� ion (m/z 283) generated from

kaempferide 3-O-glucoside mainly lost COH.

to produce

an ion at m/z 255 (Supplementary Fig. S1(c), see Supporting

Information). Nevertheless, the product ion spectrum of the

[Y0-CH3]�.

ion (m/z 284) generated from kaempferide 7-O-

rhamnoside was dominated by an ion at m/z 151, which was

attributed to the 1,3A� ion (Supplementary Fig. S1(f), see

Supporting Information).

The influence associated with the chain length of

saccharide substitutes was also investigated and the CID

spectra of kaempferide 3-O-rutinoside are shown in

Supplementary Fig. S2 (see Supporting Information). From

the spectra, no significant signal ion for the [Y0–H]�.

ion at

m/z 298 that was ascribed to homolytic cleavage was

-O-glycosides. (a) MS/MS spectrum of

of kaempferol 3-O-glucoside using the

spectrum of kaempferol 7-O-rhamnoside,

side using the [Y0–H]� ion (m/z 285) as

base peak in each spectrum.


DOI: 10.1002/rcm

Figure 2. CID spectra of kaempferol di-, tri-O-glycoside. (a)

MS/MS spectrum of kaempferol 3-O-rutinoside, (b) MS/MS

spectrum of kaempferol 3-O-kaempferol 3-O-glucosyl-(1!6)-

rhamnosyl-(1!2)-glucoside, (c) MS3 spectrum of kaempferol

3-O-glucosyl-(1!6)-rhamnosyl-(1!2)-glucoside using the

[Y0–H]� ion (m/z 285) as precursor. AI denotes the absolute

intensity of base peak in each spectrum.

Letter to the Editor 171

observed, whereas the [Y0]� ion at m/z 299 assigned to

heterolytic cleavage was the base peak. Upon CID, the [Y0]�

ion also dissociated by the loss of CH3.

to yield a radical ion at

m/z 284. Thereby, kaempferide 3-O-rutinoside displayed a

similar fragmentation behavior as kaempferide 7-O-rhamno-

side initially, which made it difficult to determine the

glycoside position simply from a comparison of their MS/

MS or MS3 spectra. Fortunately, significant differences in

their MS4 spectra between two kaempferide glycosides were

observed (Supplementary Figs. S1(f) and S2(c), see Support-

ing Information). In their MS4 experiments, both precursor

ions appeared at m/z 284. Kaempferide 3-O-rutinoside

mainly lost COH.

to generate an ion at m/z 255; whereas,

kaempferide 7-O-rhamnoside yielded an ion at m/z 256 with

similar abundance to the ion at m/z 255. Moreover, several

retrocyclization fragment ions, including the 1,3A� ion (m/z

151), appeared in the MS4 spectrum of kaempferide 7-O-

rhamnoside. Thus, the glycosylation sites of kaempferide

glycosides could be deduced and the different fragmentation

pathways between the two isomeric radical aglycone ions

Scheme 3. Proposed mechanism of f


(m/z 284) were most likely due to the different locations of

anion sites.

As discussed above, while the deprotonated kaempferol 3-

O-glucoside and kaempferide 3-O-glucoside underwent a

predominant homolytic cleavage, the deprotonated kaemp-

ferol 3-O-di-, tri-glycosides and kaempferide 3-O-di-glyco-

sides gave abundant heterolyic cleavage fragments like 7-O-

glycosides. Since the glycosylation site was the same 3-O

position, the differences in the fragmentation pathways of

kaempferol 3-O-glycosides (also the kaempferide 3-O-

glycosides) might be affected by the length of the saccharide

chains. It was proposed that CID formation of the radical

aglycone production depended on the location of the anion

and on other electron-donating substituents in the flavo-

noids. Electron donation from the substituent to the oxygen

of the active OH group weakens the O�H bond, making it

easier to release an H.

.18 The electron-donating effect of the

para-OH group substituted at the B-ring (40-OH) renders the

3-O-glycosidic bond susceptible to homolytic cleavage. It is

believed that long saccharide chains could cause large steric

hindrance and deflect the B-ring from the plane of the A-, C-

ring, thus reducing the electron-donating effect from the B-

ring. When the saccharide chains were lengthened, the

deflection was enhanced. Hence, the electron-donating effect

to the whole conjugated system from the B-ring weakens,

which makes it more difficult to release an H.

from the

flavonol glycosides. As for the kaempferol glycosides, the

relative abundance ratio of the [Y0–H]�.

ion (m/z 284) to the

[Y0]� ion (m/z 285) reduced remarkably as the substituted

saccharide chains lengthened. For kaempferide glycoside,

the CH3O group on the B-ring had larger steric bulk than

OH, so even kaempferide 3-O-di-glycoside exhibited little

homolytic cleavage.

In conclusion, we have demonstrated that the length of the

saccharide chains also plays an important role in the

fragmentation of the kaempferol and kaempferide glyco-

sides. Although homolytic cleavage is a common phenom-

enon in the fragmentation of flavonol 3-O-glycosides, a long

saccharide chain substituted at the 3-O position could hinder

the occurrence of the homolytic cleavage, resulting in similar

product ion mass spectra to those of the flavonol 7-O-

glycosides. Therefore, the previous conclusion18,23–26 that the

glycosidation site can be determined using the relative

abundance of [Y0–H]�.

ion and the [Y0]� ion in MS/MS

spectra is sometimes untenable. Nevertheless, the flavonol 7-

O-glycosides can also be differentiated from flavonol 3-O-

ormation of 1,3A� and 0,2A� ions.


DOI: 10.1002/rcm

172 Letter to the Editor

glycosides with long saccharide chains by CID of their

second-generation product ions. Therefore, for unknown

flavonoids, especially for flavonols with long saccharide

substituents, care should be taken to identify the substituted

position using the occurrence of homolytic and heterolytic

cleavage of the flavonol glycosides. Tandem mass spectrom-

etry has also proven to be a powerful tool for the structural

elucidation of the flavonol glycosides.

SUPPORTING INFORMATION

Additional supporting information may be found in the

online version of this article.

AcknowledgementsFinancial support from the National Natural Science Foundation ofChina (No. 30672600 30772721) is gratefully acknowledged.

Lin Lu1,3, Feng-Rui Song1, Rong Tsao2,Yong-Ri Jin4,Zhi-Qiang Liu1*, and Shu-Ying Liu1

1Changchun Center of Mass Spectrometry, ChangchunInstitute of Applied Chemistry, Chinese Academy of

Sciences, 5625 Renmin Street, Changchun 130022,P.R. China

2Guelph Food Research Centre, Agriculture andAgri-Food Canada, 93 Stone Road West, Guelph, Ontario,

Canada, N1G 5C93Graduate School of the Chinese Academy of Sciences,

Beijing 100039, P.R. China4Jilin University, College Chemistry, Changchun 130023, P.R.

China

*Correspondence to: Z.-Q. Liu, Changchun Center of Mass Spectro-metry, Changchun Institute of Applied Chemistry, Chinese Acad-emy of Sciences, Changchun 130022, P.R. China.E-mail: [email protected]

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Received 23 September 2009Revised 4 November 2009

Accepted 5 November 2009


DOI: 10.1002/rcm

studies on the homolytic and heterolytic cleavage of kaempferol and kaempferide glycosides using...

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