Mn2+

O

OOH

HO

OHOH

OO

O

OHHO

OH

Mn2+

O

OOH

HO

OH

OH

OO

HO

O

OH O

H

Mn2+

O

OOH

HO

OH

OH

OOH

O

O

OH

O

H

Saccharide Characterization of Monoglycosyl Flavonoids By LC-MSn with Post-Column Manganese Complexation

Barry D. Davis, Jennifer S. BrodbeltThe University of Texas at Austin, Department of Chemistry and Biochemistry, Austin TX 78712

OVERVIEW

INTRODUCTION

There is a long history of using mass spectrometry for flavonoid identification.1,2 However, determining the identities and locations (see Figure 1 for standard numbering scheme) of attached saccharide moieties remains a considerable challenge. Only one mass spectrometric method for identifying isomeric monosaccharide moieties (i.e. glucose vs. galactose) of flavonoid glycosides has been published, but the method requires isolation and an overnight derivatization of the analytes prior to analysis.3 In this work, a metal complexation strategy is successfully applied to on-line analysis of extracts from the peel of Fuji apples and the flesh of red onions via post-column addition of MnCl2. Dissociation of the manganese complexes provides information on the identities and locations of the saccharide moieties.

1. Cuyckens, F.; Claeys, M. J. Mass Spectrom. 2004, 39, 1-15.2. Stobiecki, M. Phytochemistry 2000, 54, 237-256.3. Cuyckens, F.; Shahat, A. A.; Pieters, L.; Claeys, M. J. Mass Spectrom.

2002, 37, 1272-1279.4. Dick, A. J.; Redden, P. R.; Demarco, A. C.; Lidster, P. D.; Grindley, T. B.

J. Agric. Food Chem. 1987, 35, 529-531.5. Marotti, M.; Piccaglia, R. J. Food Sci. 2002, 67, 1229-1232.6. Gaucher, S. P.; Leary, J. A. Anal. Chem. 1998, 70, 3009-3014.7. Scheiber, A.; Keller, P.; Streker, P.; Klaiber, I.; Carle, R. Phytochem.

Anal. 2002, 13, 87-94.8. Sánchez-Rabaneda, F.; Jáuregui, O.; Lamuela-Raventós, R. M.;

Viladomat, F.; Bastida, J.; Codina, C. Rapid Commun. Mass Spectrom.2004, 18, 553-563.

9. Lommen, A.; Godejohann, M.; Venema, D. P.; Hollman, P. C. H.; Spraul, M. Anal. Chem. 2000, 72, 1793-1797.

10.Oleszek, W.; Lee, C. Y.; Jaworski, A. W.; Price, K. R. J. Agric. Food Chem. 1998, 36, 430-432.

11.Day, A. J.; Mellon, F.; Barron, D.; Sarrazin, G.; Morgan, M. R. A.; Williamson, G. Free Radical Res. 2001, 35, 941-952.

12.Park, Y. K.; Lee, C. Y. J. Agric. Food Chem. 1996, 44, 34-36.13.Ferreres, F.; Gil, M. I.; Tomás-Barberán, F. A. Food Res. Int. 1996, 29,

389-395.14.Tsushida, T.; Suzuki, M. J. Jpn. Soc. Food Sci. 1995, 42, 100-108.

ResultsØ Characteristic fragments for glucosides, galactosides, arabinosides, and xylosides were found

Ø The position of glycosylation may be determined using Mn(II) complexation

Ø Flavonoid glycosides in the extracts were positively identified without the need for complementary or supplemental analytical methods

Flavonoid glycosides in the extracts were positively identified without the need for further anaylsisFollowing the discovery of these diagnostic dissociation pathways, several monoglycosyl flavonoids were identified directly from extracts of Fuji apples and red onions by LC-MSn analysis, with the Mn(II) complexes formed on-line via post-column addition of MnCl2. Apples were a special target of interest because they are known to contain several monoglycosyl flavonoid isomers that differ only by the identity of the saccharide moieties. The flavonoids in the peel were extracted with methanol4 and selectively eluted by solid phase extraction. The UV chromatogram (Figure 5) shows at least 8 flavonoid components in the extract, along with some early-eluting cinnamic acids. Negative ion mode tandem mass spectrometry was first employed to obtain the molecular weight of each compound, along with the aglycon and saccharide weights. Compounds 1-7 each lost a single saccharide moiety, leaving behind a deprotonated aglycon with mass 301. Further fragmentation proved that this aglycon was quercetin in all cases. (8 was later determined to be a member of the chalcone family, a minor flavonoid class that is outside the scope of the current study.) In order to probe the identities and locations of the saccharides, post-column Mncomplexation was employed in the positive ion mode. The following compounds were identified based on the CAD data from their Mn complexes: 1 – quercetin-3-O-galactoside; 2 – quercetin-3-O-glucoside; 3 – quercetin-3-O-xyloside; 5 – quercetin-3-O-arabinofuranoside; 7 – quercetin-3-O-rhamnoside (based on mass of the saccharide). 4 and 6 did not give sufficiently abundant complexes for further analysis.

A similar analysis was performed on an extract from red onion. The flavonoids were extracted with a methanol/water/acetic acid mixture5 and selectively eluted by solid phase extraction. The UV chromatogram shows at least 7 flavonoid components (Figure 6). Negative ion mode proved that these were a mixture of monoglycosides and diglycosides, with aglycons of two different molecular weights. The aglycons were determined to be quercetin (m/z 301) and isorhamnetin (m/z 315) based on their fragment ions. Compound 15 was simply the aglyconquercetin. The following compounds were identified based on the CAD data from their Mncomplexes: 12 – quercetin-3-O-glucoside; 13 – quercetin-4'-O-hexoside; 14 – isorhamnetin-4'-O-hexoside. CAD energies were used as confirmational evidence for differentiating 3-O and 4'-O glycosides. The diglycosides are outside the scope of this study, but at least one of the hexosesof 10 was determined to be at the 4' position on the quercetin molecule.

Several methods were used to confirm the compound identifications made by Mn complexation. Retention time matching with standards confirmedthe identities of 1, 2, 3, 5, 7, 12 and 15. The saccharide moiety of 13 was concluded to be glucose based on retention time matching. No standard for isorhamnetin-4'-O-glucoside was available for comparison to 14, but an isorhamnetin-3-O-glucoside standard eluted earlier than 14, which is consistent with our observation that 3-O-glycosides generally elute earlier than 4'-O-glycosides. The remaining compounds are all outside the scope of this study, and their identities were inferred from previous studies of apple and onion flavonoids.4,5,7-14 This yields the following tentative identifications: 4 – quercetin-3-O-arabinopyranoside; 8 – phloretin-2'-O-xyloglucoside; 9 – quercetin-7,4'-di-O-glucoside; 10 – quercetin-3,4'-di-O-glucoside; 11 – isorhamnetin-3,4'-di-O-glucoside. 6 has been observed previously but has defied attempts to fully characterize it, including NMR spectroscopy.9

REFERENCES

MnCl2, 500 µM20 µL/min

C18 column 2.1 x 50 mm mixing teeHPLC

H2O/ACN0.33% formic acid0.3 mL/min

ESI interface+4.5 kV

Quadrupole ion trap

Full scan mass spectraCollisional activated dissociation (CAD)

Ion optics

O

OOH

HO

OHGlc

O

OOH

GlcO

OH

O

OOH

GlcO

OHOH

O

OOH

HO

OGlcOH

OH

O

OOH

HO

OH

Glc

O

OOH

GlcO

OH

O

OOH

HO

OHOH

OXyl

O

OOH

HO

OHOH

OAra

O

OOH

HO

OGlcOH

O

OOH

HO

OH

OGlc

O

OOH

HO

OHOH

GlcO

OOH

HO

OHOH

Glc

O

OOH

GlcO

OHOH

OH

O

OOH

HO

OHOH

OGal

O

OOH

HO

OHOH

OGlc

O

OOH

HO

OHOCH3

OGal

OCH3O

OOH

HO

OHOCH3

OGlc

OCH3O

OOH

HO

OHOCH3

OGlc

Oflavonoidaglycon

HOHO

OH

O

OH

flavonoidaglycon

HO

HO

OHO

flavonoidaglycon

HO

OH

OH

Oflavonoidaglycon

HOOH

OHOH

apigenin-7-O-glucosideMW=432flavone

apigenin-6-C-glucoside(isovitexin)MW=432 flavone

apigenin-8-C-glucoside(vitexin)MW=432flavone

quercetin-3-O-arabinofuranoside(avicularin)MW=434 flavonol

quercetin-3-O-xyloside(reynoutrin)MW=434 flavonol

naringenin-7-O-glucoside(prunin)MW=434 flavanone

kaempferol-3-O-glucoside(astragalin)MW=448 flavone

luteolin-6-C-glucoside(homoorientin)MW=448 flavone

luteolin-8-C-glucoside(orientin)MW=448flavone

quercetin-4'-O-glucoside(spiraeoside)MW=464 flavonol

luteolin-4'-O-glucosideMW=448 flavone

luteolin-7-O-glucosideMW=448 flavone

quercetin-3-O-galactoside(hyperoside)MW=464 flavonol

quercetin-7-O-glucosideMW=464 flavonol

quercetin-3-O-glucosideMW=464 flavonol

syringetin-3-O-glucosideMW=508 flavonol

syringetin-3-O-galactosideMW=508 flavonol

Glc=ß-D-glucopyranosyl

Gal=ß-D-galactopyranosyl

Ara=a-L-arabinofuranosyl

Xyl=ß-D-xylopyranosyl

MethodsØ Formation of flavonoid glycoside/Mn(II) complexes for structural characterization using collisional activated

dissociation (CAD)

Ø Analysis of apple and onion extracts by LC-MSn with post-column addition of MnCl2

PurposeØ To find a simple LC-MS method for identifying saccharide moieties of monoglycosyl flavonoids

isorhamnetin-3-O-glucosideMW=478 flavonol

ACKNOWLEDGEMENTS

This material is based upon work supported under a National Science Foundation Graduate Research Fellowship to BDD. This work is also supported by the National Institutes of Health (NIH RO1 GM63512) and the Welch Foundation (F-1155).

CONCLUSIONS

Ø CAD using flavonoid glycoside/Mn complexes of the form [Mn(II) (L) (L-H)]+ and [Mn(II) (L)2 (L-H)]+ are useful in characterizing the saccharide portions of monoglycosyl flavonoids

Ø In the case of flavonoid hexosides, the most common glycosylation positions (3-O, 4'-O, 7-O, 6-C and 8-C) may be determined

Ø In the case of flavonoid-3-O-glycosides, an effective method for differentiating glucose and galactose was found. A xyloside and an arabinofuranoside have also been differentiated

Ø Mn complexation is easily applied to on-line LC-MS analysis; the method may therefore be applied directly to complex mixtures without first isolating individual components

Ø Supporting evidence for compound identifications may be obtained from retention time comparison with authentic standards, knowledge of the elution order associated with various structural features, and confirming the characteristic CAD energies needed to dissociate various complexes

Ø Compound identifications made by this method are in agreement with the literature

METHODS

Ø For direct infusion studies, flavonoid standards (Figure 2) and MnCl2 were dissolved in methanol, 10 µM each

Ø A Finnigan LCQ Duo quadrupole ion trap mass spectrometer with electrospray ionization (ESI) was used

Ø CAD was used to find fragmentation pathways characteristic of analyte structure

Ø Flavonoids were extracted4,5 from Fuji apple peel and from red onions

Ø Extracts were separated by HPLC, with MnCl2 added post-column, and the mixture was fed to the ion trap mass spectrometer for analysis (Figure 3)

RESULTS

Characteristic fragments for glucosides, galactosides, arabinosides, and xylosides were foundDifferentiation of flavonoid 3-O-hexosides (glucosides and galactosides) is observed by MS3

fragmentation of the 2:1 flavonoid glycoside/Mn complex, [Mn(II) (L) (L-H)]+, where L is the flavonoid glycoside. When these complexes are subjected to CAD, the only fragment ion results from the loss of one hexose moiety, -162 u. However, performing a second stage of CAD on this key primary fragment ion leads to a clear differentiation of the 3-O-glucosides and -galactosides. All of the complexes exhibit losses of the second hexose moiety and of one aglycon unit, but the flavonoid galactoside complexes display the additional loss of an aglycon plus 102 u, distinguishing these compounds from the corresponding flavonoid glucosides (Figure 4). The loss of 102 u from flavonoid galactosidecomplexes is theorized based on a similar fragmentation observed by Gaucher and Leary from hexose/Zn(II)/dien complexes6 (Scheme 1). A similar method employing the 3:1 complex, [Mn(II) (L)2(L-H)]+, was found to differentiate quercetin-3-O-arabinofuranoside and quercetin-3-O-xyloside. Using single-stage CAD, the arabinoside complex yields only one significant product ion stemming from the loss of one flavonoid glycoside. However the xyloside complex also yields an abundant fragment ion corresponding to the loss of a flavonoid glycoside plus a pentose moiety (Figure 4).

The position of glycosylation may be determined using Mn(II) complexationIn addition to allowing the confident identification of the saccharide moiety, the Mn(II) complexes provide a means to determine the glycosylation sites of the flavonoid hexosides. Unique and consistent fragmentation patterns are observed for complexes involving flavonoids glycosylated at five common positions: attachment through an oxygen atom at position 3, 4' or 7; or through a carbon atom at position 6 or 8. The MS/MS fragments yielded from the [Mn(II) (L) (L-H)]+ complexes are summarized in Table 1. For each glycosylation site, a specific set of fragment ions from the complex is obtained. The loss of a hexose residue is indicative of O-glycosylation. The 3-O-hexoside complexes yield no other significant fragments, while the 4'-O-hexosides also show several additional fragments ions. The 7-O-hexoside complexes are the only ones to display both the loss of a hexoseresidue and a 0,2 cross-ring saccharide cleavage (-120 u). This cross-ring cleavage is the most abundant fragment ion for the C-glycoside complexes. 6-C and 8-C glycosides can be differentiated by other fragment ions. The CAD energies are also characteristic of the flavonoid glycosylation site.

150 5 10 20 25min

UV

280

inte

nsity

1

2

3

4

5

6/7 8

1: 463 301 (aglycon)

2: 463 301 (aglycon)

3: 433 301 (aglycon)

4: 433 301 (aglycon)

5: 433 301 (aglycon)

6: 433 301 (aglycon)

7: 447 301 (aglycon)

8: 567 273 (aglycon)

-162

-162

-132

-132

-132

-132

-146

-(132+162)

150 5 10 20 25 30

10

13

14119 12 15

min

UV

280

inte

nsity

9: 625 463 301 (aglycon)

10: 625 463 301 (aglycon)

11: 639 477 315 (aglycon)

12: 463 301 (aglycon)

13: 463 301 (aglycon)

14: 477 315 (aglycon)

15: 301 (aglycon)

-162

-162

-162

-162

-162

-162 -162

-162 -162

Figure 2. Flavonoid glycosides included in this study

Figure 3. Instrumental set-up for LC-MSn with post-column complexation Figure 4. CAD spectra for selected flavonoid glycoside/Mn(II) complexes

Scheme 1. Proposed mechanism for loss of 102 u from flavonoid galactoside complexes

Table 1. Relative abundances of selected fragment ions from [Mn(II) (L) (L-H)]+ complexes (N = 2-3)

–

–

–

–

100

100

100

100

100

100

100

100

100

100

100

100

-Hex

–

–

–

–

12

10

15

8

5

10

–

–

–

–

–

–

-2 Hex

–

–

–

–

2

2

3

2

8

7

–

–

–

–

–

–

-A

100

100

100

100

11

13

6

14

–

–

–

–

–

–

–

–

-120

2

3

40

40

3

2

–

2

–

–

–

–

–

–

–

–

-H2O

–

–

14

15

–

–

–

–

–

–

–

–

–

–

–

–

-(120 & H2O)

7

6

4

4

–

–

–

–

–

–

–

–

–

–

–

–

-90

448

432

448

432

464

448

434

432

464

448

508

508

478

464

464

448

MW

19.9luteolin-8-Glc

20.3apigenin-8-Glc8-C

22.6luteolin-6-Glc

23.0apigenin-6-Glc6-C

22.2quercetin-7-Glc

24.3luteolin-7-Glc

20.7naringenin-7-Glc

23.9apigenin-7-Glc

7-O

22.4quercetin-4'-Glc

25.9luteolin-4'-Glc4'-O

18.0syringetin-3-Gal

17.6syringetin-3-Glc

17.9isorhamnetin-3-Glc

18.3quercetin-3-Gal

17.8quercetin-3-Glc

18.8kaempferol-3-Glc

3-O

CAD%

Flavonoid Glycoside

Glycos. Site

Figure 5. UV chromatogram of apple peel extract, with summary of saccharide losses

Figure 6. UV chromatogram of onion extract, with summary of saccharide losses

A: H2O, 0.33% formic acidB: ACN, 0.33% formic acidIsocratic @ 12% B, 0.3 mL/min10 µL injection

Negative modeMS/MS results

Negative mode MS/MS results

A: H2O, 0.33% formic acidB: ACN, 0.33% formic acid10% B to 25% B in 10 min, 0.3 mL/min10 µL injection, 10x dilution

Abbreviations: Hex = hexose moiety, A = aglycon portion, 120 = 0,2 saccharide cleavage, 90 = 0,3 saccharide cleavage.CAD energies were selected so that the parent ion was reduced to 5-10% relative intensity.

isorhamnetin-3-O-glucoside

kaempferol-3-O-glucoside

syringetin-3-O-galactoside

syringetin-3-O-glucoside

quercetin-3-O-galactoside

quercetin-3-O-glucoside

746-Hex

746-Hex

562-A

562-A

518-A

518-A

658-Hex

658-Hex

626-Hex

686-Hex532

-A

502-A

460-(A+102)

416-(A+102)

500300 400 600 700 800 900

820

*

820

*

908

*

908

*

788

*

848

*

m/z

100

100

100

100

100

100

0

0

0

0

0

0

quercetin-3-O-xyloside

600 1400800 1000 1200

922-L790

-(L+Pent) 1224-Pent

11392+-L

quercetin-3-O-arabinofuranoside922-L

1356

*

1356

*

100

1000

0

m/z

Spectra of flavonoid hexoside complexes are the MS3 from [Mn(II) (L) (L-H)]+ following the loss of one hexose moiety, with CAD energy 22-23%. Spectra of flavonoid pentoside complexes are the MS/MS from [Mn(II) (L)2 (L-H)]+ with CAD energy 17%. The parent ions are denoted by an asterisk (*). Fragment ions are labeled as follows: -Hex (loss of a hexose moiety); -A (loss of an aglycon portion); -L (loss of a flavonoid glycoside); -Pent (loss of a pentose moiety); 2+-L ([2 Mn(II) (L)4 (L-H)2]2+ - L).

O

O

3

7

56

8

3'4'

5'6'

2'

4

2 1'

Figure 1. Standard numbering scheme for carbon atoms of flavones and flavonols

-102 u,C4H6O3

--

-