ADV06-15_1-412

Foods, Flavors

Fragrances

Rapid, Reproducible HPLC Analysis for Flavonoids in Cocoa

Using a LECO Unique" LC-TOFMS System and an Ultra Aqueous C18 Column

ByJulie Kowalski, Restek Innovations Chemist, and Brian Shofran, LECO Corporation

• 15-minute screening for flavonoids.

• Excellent select ivity, using an

Ultr a Aqueou s C18 column.

• Reliabl e identifications and reproducib le

results for complex samples.

Flavonoids arc complex polyphenolic compounds,

with diverse aromatic sub stitutions , that con

tribute to color, flavor, fragrance-and toxicity

of many food s. Interest in flavonoid s has exploded

becau se of links to antioxidant activity and, possi

bly, to control and prevention of disease.':'

Flavonoid contents of foods have been difficult to

study, due to sample complexity and generally low

abundances of the target compounds. Cocoa is

rich in th e flavan- 3-ol flavonoids, including cate

chin, cpicatechin, and procyanidin (Figure I), and

these are screened for as marker compounds. In

finished choco late and cocoa products, amounts of

flavonoids depend primarily on the amounts of

nonfat cocoa solids, on bean typ e, and on process

ing. Flavonoids can be destroyed by heat or oth er

processing, like dutching, which is common in the

production of cocoa and chocolate products.

We developed a rapid screening met hod for cate

chin, epicat echin , and procyanid in content, and

screened commercial cocoa products for flavan-3

ol content. We pr epared samples by mixing the

cocoa products with liquid nitrogen, powdering

the frozen mixes, and extracting samples wit h

deionized water : methanol (1:4). Extracts were

Figure 1

Flavan-3-ol flavono ids are

screened for as marker compounds.

-c

"" I

catechin

epicatechin

procyanidin dimmer

Procyanidin oligomers

1-10)

2006.03

Figure 2

Extracted ion chromatogram of a cacao sample.

20000

18000

16000

14000

12000

10000

8000

6000

4000

2000

1:40

Conditions:

Mobilephase:

Flow:

Temp.:

Del.:

Mass Spectrometry

Instrument

ESI voltage:

Desolv. temp.:

Nebulizer pres.:

Desolv. gas:

Interfacetemp.:

Nozzle:

Data

acq.

rate:

7 9 1011121516.17J9jiU1 24

4 \ \

1////~/

sample:

Inj.:

5/1l

Cone.:

500mgsample extract

Sample diluent:

70%water/methanol

Autosampler temp: I O' C

Column:

Ultra Aqueous C18

Cat.# :

9178312

Dimensions:

100x 2.1mm

Particlesize:

3/1111

Pore size:

100A

3:20 5:00 6:40

8:20 10:00 11:40 13:20 15:00 Time (mm:ss)

lCJF0405

A: 0.1%formic acid inwater; B: acetonitrile:methanol,50:50(v/v)

Time(min.)

Numbered peaks are

400/1l / min.

listed in Table 1

30'C

UV @ 210nm

lecoUnique' l C-TOFMSHighFlow ESISource

(.) 3500V

300'C

375kPa

nitrogen, 7L/ min.

100' C

(-)160V

4spectra/ sec.

Table 1

Components in the cacao sample.

Peak

RT (min:sec

_ -"Un""iljue Mass

4. catechin (monomer)

03:50.4

289.1818

L Q!Q9'anidin

B~2~

--:0!:.:4!.:!2,~4___

S77.3722

icatech

04:53.8

289.1841

!Q,J>rocyanidin

05:06.2

865.5671

11. procyanidin l!etramer)

05:17.8

11_~~JI7L9

12. clovamide

05:29.3

358.2409

li.....!Jrocyanidin II-9.

06:21.1

737.4785

!LProcyanidin B5

06:31.7

577.3745

1L.Qroc anidin II-a

06:32.6

707.4643

19. dideoxyclovamide

07:08.2

326.2384

20. quercetin- alactoside

07:16.8

463.279

Area

28618

6.7

3 S9

8.0

~:4~5~~

--,~c-

93682

21.8

10221

2.4

~5 5

0.4 _

::8::-

---,~

3528

0.8

5246

1.2

10339

2.4

4043

0.9

4839

1.1

9471

2.2

21. quercetin-arabinoside

07:44.6

433.2524

9797

2.3

~quercet

Identities of peaks

1,2,3,5,6,8,13,14,18,22,23,25,26

areunknown,but retention

times, masses, areas, and area

areavailable onrequest, and

will belistedinournext Buzz

electronic newsletter.

09}0.2

3Q1J595

2179

0.5__

Website :

www.chromtech.net.au

E-mail :

info@chromatech.net.au

TelNo : 03 9762 2034 . . . in AUSTRALIA