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Application Note No 0002

Research on Betalain Pigments from Red Beetroot (Beta vulgaris) Extracts Using LC-DAD-ESI-MS/MS system (LCMS-8030 Shimadzu)

INTRODUCTION

Betalains are water-soluble plant pigments

recently emerging as very valuable health-

promoting antioxidants (Cai at all. 2003, Gandia-

Herrero at all. 2010, Wybraniec at all. 2010). Many

betalains, such as betanin Fig. 1, are 5-O-glucosides

of betanidin (the basic chromophoric aglycone

unit). Betanin colorant (E-162) is produced from

red beetroots (Beta vulgaris L.) and is available as a

concentrate produced by vacuum evaporation of

beet juice, or as a powder made by spray-drying of

the concentrate (Nemzer at all. 2011).

Fig. 1. Chemical structure of betanin, one of the most fre-

quently occuring pigments in Beta vulgaris L. root. The long chromo-

phoric unit is marked in purple.

EQUIPMENT AND METHODS

Analysis of different betalain pigments

from Beta vulgaris L. root extracts was performed

by high-performance liquid chromatography

equipped with a diode array detection and tandem

mass spectrometry with electrospray ionization

(LC-DAD-ESI-MS/MS). This work presents a pro-

cedure which enables a simultaneous analysis of

betalain pigments using negative and positive

modes. Among all the available methods for the

analysis of betalain pigments, reversed-phase high-

Fig. 2. UHPLC Nexera XR Shimadzu. SIL-20ACXR autosampler

equipped with MTP holder (Photo: P. Stalica).

performance liquid chromatography (RP-HPLC)

coupled with DAD and MS/MS is the most popular

choice.

HPLC CONDITIONS

The chromatographic separation was car-

ried out using an HPLC system (Shimadzu, Japan)

consisting of a degasser DGU-20A3R, controller

CBM-20A, binary pump Nexera XR LC-20 ACXR,

autosampler Nexera XR SIL-20ADXR and column

oven CTO-20AC. The analytes were separated on a

Kinetex C18 column, 100 mm×4.6 mm, 5 µm (Phe-

nomenex). The temperature of the column oven was

set to 40 °C, the flow rate was





kept at 0.5 mL/min and the injection volume was

set from 1 to 20 µL. The mobile phase used for the

separation was 2% formic acid in water (component

A) and MeOH (component B). The chromatograph-

ic separation was performed in gradient elution

mode: 0 min (5 % B), 12 min (25 % B), 15 min (70

% B). The total time of the chromatographic run

was 16 min, while the column equilibration time

was set to 5 min. The chromatogram presenting the

separation of analytes is shown in Fig. 4.

MS/MS CONDITIONS

All analyses were done using a Shimadzu

LCMS-8030 triple quadrupole mass spectrometer

(Shimadzu, Japan) equipped with an ESI source

working in the polarity switching MRM mode, scan

mode and mainly in the Product Ion Scan mode.

Data acquisition and analysis were accomplished

with LabSolutions 5.60 SP1 software.

Fig 3. LCMS-8030 belongs to the series Ultra-Fast Mass Spectrometers from Shimadzu, Japan

Fig. 4. Contour plot of LC separation of the pigments from the aqueous

extract from Beta vulgaris L. root.

Fig. 5. UV-VIS spectrum of betanin (λmax=534 nm) obtained in the

eluent during the HPLC run.

Fig. 6. Contour plot (zoom) of LC separation of the pigments in the

aqueous extract from Beta vulgaris L. roots. The dominant compounds

are betanin (Rt=5.3 min), vulgaxanthin I (Rt=3.4 min) and isobetanin

(Rt=6.1 min).

Fig. 7. 3D plot of LC separation of betalains in the aqueous extract

from Beta vulgaris L. root.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

200

300

400

500

600

700

800

nm

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 min

300

400

500

600

nm

200 250 300 350 400 450 500 550 600 650 700 750 nm

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

1400

1500mAU

Betanin





Fig. 8. ESI-MS spectrum of betanin detected by LC-ESI-MS analysis

in positive mode (m/z = 551)

Fig. 9. ESI-MS spectrum of betanin detected by LC-ESI-MS

analysis in negative mode (m/z = 549)

Fig. 10. ESI-MS spectrum of 17-decarboxy-betanin detected

by LC-ESI-MS analysis in positive mode (m/z = 507).

Fig. 11. ESI-MS spectrum of 17-decarboxy-betanin detected

by LC-ESI-MS analysis in negative mode (m/z = 505).

Fig. 12. ESI-MS chromatogram of betanin, isobetanin and its decarboxylated compounds detected in positive mode.

542 545 547 550 552 555 7 560 m/z0.0

1.0

2.0

3.0

(x1,000,000)551.00

552.00

Betanin

542 45 547 550 552 555 557 560 m/z0.0

1.0

2.0

3.0

4.0

Inten.(x100,000)

549.00

550.00

Betanin

504 505 506 507 508 509 510 511 m/z0.0

0.2

0.5

0.7

1.0

1.2

1.5

Inten. (x1,000,000)507.00

508.00

17-decarboxy-betanin

504 505 506 507 508 509 510 511 m/z0.00.20.50.71.01.21.51.72.0

Inten.(x100,000)

505.00

506.05

17-decarboxy-betanin

3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 min0

2500000

5000000

7500000

10000000 1:507.00(+)1:551.00(+) Betanin

Isobetanin

17-decarboxybetanin 17-decarboxyisobetanin

15-decarboxybetanin

OH N+

H

NH

15

O

OHOH

OH

OH

O

COOH

COOH+

OH N+

2

NH

O

OHOH

OH

OH

O

COOHHOOC

+COOH

+

OH N

HO

OHOH

OH

OH

O

17NH

HOOC

+

OH N

2

O

OHOH

OH

OH

O

17NH

HOOC

A B C D

2-decarboxy-

betanin 15-decarboxy-

betanin

17-decarboxy-

betanin 2,17-decarboxy-

betanin

Fig. 13. Structures of decarboxylated derivatives of betanin pigment. Position of decarboxylation has an influence on color

and antioxidant activity.





Fig. 14. LC-DAD contour plot of betanine, isobetanin and its decarboxylated compounds

Fig. 15. ESI-MS spectrum of betanin in positive mode before frag-

mentation (m/z = 551) and after fragmentation in Collision Cell (m/z

= 388)

Fig. 16. Fragmentation spectrum of decarboxylated betanin in posi-

tive mode (precursor ion m/z 507)

Fig. 17. Fifteen betalain pigments detected in redbeet juice

RESULTS AND DISCUSSION

We obtained interesting results using the

LC-DAD-MS/MS system from Shimadzu. Fig. 4,

5, 6, 7 show DAD chromatograms of Beta vulgar-

is L. root extract. Fig. 17 additionally shows the

MS chromatograms of 15 betalain pigments which

are naturally occurring in plants. The analysis of

pigments not only included the naturally occurring

pigments, but also the analysis on the thermal

stability of the discussed compounds. Fig. 10, 11,

12 present the spectra of the degradation products

of betanin. Fig. 14 shows the DAD chromatogram

of decarboxylated derivatives of betanin. These

are the most frequently occurring pigments which

are initially found as the first derivatives on the

degradation pathway. These include the 17-

decarboxylated compounds, 15-decarboxylated

compounds and 2-decarboxylated compounds

(Fig. 13). The fast polarity switching capability of

the Shimadzu LCMS-8030 MS/MS detector cou-

pled with the Nexera UHPLC system were crucial

in achieving the analytical results and developing

this application

LITERATURE

1. Cai Y, Sun M i Corke H (2003) J Agric Food Chem

51: 2288

2. Nemzer B, Pietrzkowski Z, Spórna A, Stalica P,

Thresher W, Michałowski T, Wybraniec S (2011)

Food Chemistry 127(1): 42

3. Gandia-Herrero F, Escribano J, Garcia-Carmona F

(2010) Planta 232(2): 449

4. Wybraniec S, Stalica P, Spórna A, Mizrahi Y

(2010) J Agric Food Chem 58(9): 5347

ACKNOWLEDGMENTS

The autor would like to thank dr. hab.

Sławomir Wybraniec from Cracow

University of Technology, Institute C-

1for providing the results and scientific

consultation.

9.0 10.0 11.0 12.0 13.0 14.0 min

450

500

550

600

nm

450 500 550 600 650 700 750 800 850 m/z 0.0

2.5

5.0

Inten. (x1,000,000)

551.00

507.00 417.00 889.00

100 150 200 250 300 350 400 450 500 m/z 0.0

2.5

5.0

7.5

Inten. (x100,000)

388.90

100 150 200 250 300 350 400 450 500 m/z 0.0

2.5

5.0

7.5

Inten.(x1,000)

344.85

149.95 238.20107.50

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min

55

0.0

0.5

1.0

1.5

2.0

2.5

(x1,000,000)

8:503 TIC(+) CE: -

35.0Inten.

7:549 TIC(+) CE: -35.06:505 TIC(+) CE: -35.05:507 TIC(+) CE: -35.04:Bt i IsoBt TIC(+) CE: -35.0 3:341 TIC(+) CE: -35.02:360 TIC(+) CE: -35.01:461 TIC(+) CE: -35.0

6.953 17.463 11.910 17.963 8.445 9.815

5.691

17.314

5.697

6.403

3.740 4.110516.705 17.209

3.732 10.276

17-dBt 17-dIBt 15-dBt 2-dBt

Bt IBt

nota aplikacyjna betalain pigments poprawki sw last high-performance liquid chromatography equipped...

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