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Foods, Flavors & Fragrances
Figure 3 The flavonoid composition of cocoa powder is readily distin­
gu ished from that of cacao, using our column and detection system.
59 11131718 20
20000
18000
16000
14000
12000
1000
8000
6000
4000
2000
Conditions:
Mobile phase:
Flow:
Ternp.:
Del.:
<,<, I ...... ,
1:40 3:20 5:00 6:40
'"
centrifuged, concentrated , and filtered.' For a
detai led description of sample preparation, refer to
the LECO website .
An Ultra Aqueous C IS column is an excellent
choice for this analysis, because it is designed to
perform rever sed phase separations well and
reproducibl y when the mobile phase has a high
aqueous con tent. Using a IOOmm x 2.Imm Ultr a
Aqueous C IS column and the automated peak
find LECO ChromaTOF software in the Unique's
LC-TO FMS system , we separated and identified 26
flavonoid compounds in a cacao sample (Figure 2
and Table 1).*
Next, using the automated peak find software in
ChromaTOF, we identified flavonoids in cocoa
powder (Figure 3 and Table 2). Processing of cacao
reduces th e amount of catechins and procyanidins
in cocoa components. If an alkalizing step is pres­
ent in the process, this also leads to a remarkable
decrea se in the content of catechins and procyani­
din s. For peaks identified in the cocao and cocoa
powder samples, retention time did not differ by
more than 0.01 seconds (Tables 1 and 2). The
ana lysis was completed and conditions returned to
the initial mobil e phase composition in 15 min utes.
Subsequently, we ana lyzed three samples from
Venezuela, containing differing amounts of cacao.
Quantitative results were determined through
ChromaTOF. Analytical results for these samples are
shown in Figure4.As expected, based on data in Table
1, epicatechin was substantially higher than catechin
in each sample. Also as expected, catechin, epicate­
chin, and procyanidin B2 content increased with
increasing amounts of cacao.
A LECO Unique's LC-TOFMS system and an Ultra
Aqueous C IS column assure rapid, excellent resolu­
tion, reliable identification and quantification, and
Sample:
Inj.:
Cone.:
Sample diluent:
Autosarnpler temp:
Column:
Cal.# :
Dimensions:
Particlesize:
Pore size:
51ll
500 mg sample extract
70%water/ methanol
10'C
Ultra Aqueous C18
9178312
100 x 2.1rnrn
311"1
100A
8:20 10:00 11:40 13:20 15:00 Time(mm:ss)
LCJ F0406
A: 0.1%formic acid inwater; B: acetonitrile:methanol, 50:50 (v/ v)
Time (min.) %B
o 10
10 60
15 60 Numbered peaks are
400lll /min. listed in Table 2
30'C
UV @ 210nm
MassSpectrometry
Instrument leco unique" l C-TOFMSHigh Flow ESI Source
ESI voltage: (-) 3500 V
Desolv. temp.: 300' C
Nebulizer pres.: 375kPa
Desolv. gas: nitrogen, 7L1min.
Interface temp.: 100'C
Nozzle: (-) 160V
Data acq, rate: 4 spectra/sec.
Table 2 Flavono id components in cocoa powder exhibit virtually
the same retention times as in cacao.
Peak RT min:sec UnigueMass Area Area %
5. catechin (monomer) 03:50.4 289.1806 35151 8.7
9. procyanidin B2 04:25.0 577.3661 3928 1.0
11. epicatechin 04:52.8 289.1802 28030 6.9
13. procyanidin Cl 05:28.3 358.2432 3287 0.8
17. procyanidin (tetramer) 07:08.2 326.2279 7088 1.8
07:16.8 463.2485 6002 1.5
18. c1ova"'m"'id":e:-::=-- -"':'~~---'="="---------"'''''''----~-- highly reproducible reten tion times for flavonoid
07:43.7 433.2532 6047 1.5
~LQ9'an i i Il-g ==''''-__ ---'~'__ d n -"-''-'2::''-'- ~'___
compounds - even in very complex mixtures.
Identitiesof peaks 1,2,3,4,6,7,8,10,12,14,15,16,19,21,22are unknown,but retentiontimes, masses,areas, and
area %are availableonrequest, andwill be listed inournext Buzzelectronic newsletter.
References
Figure 4 Concentrations of flavonoids in Venezuelan cacao,
determined using an Ultra Aqueous C18 column and a
LECO Unique " LC-TOFMS system.
12 ,-- - - - - - - - - - - - - - - - - - - - - - - - - --,
~ 10
_ _ catechin
.;l" 81-- - - - - - - - - - - - - - . epicatechin
0'1 procyanidin B2
.5 6 1-- - - - - - - - - - - - - - - - - - - ___.
• 25 •
1. Prior, R.L., et al., Procyanidin and catechin content andantioxidant
capacity of cocoa andchocolate products, J. Agric. FoodChem. 54:
4057-4061 (2006).
2. Hurst,W.J., et al., Antioxidantactivity andpolyphenols andprocyani­
din contentsof selectedcommercially availablecocoa-containing and
chocolate products in the UnitedStates, J. Agric. Food Chem. 54:
4062-4068 (2006).
3. Andreas-l acueva, et al., An LCmethod for theanalysis of cocoa phe­
nolics, LC*GCf ur. 902-905(2000).
l ECO Corporation, 149S0Technology Court, Fort Myers, Fl, 33912
*Cacao is the sumof the products derived fromthe cacao bean­
chocolate liquor, cocoa, and cocoa butter',
Ultra Aqueous C18, 5~m Column
~m Column 2.1mm cal # rice
100mm 9178312 $370
800-356-1688 •
Website : E-mail : TelNo : 03 9762 2034 . . . in AUSTRALIA