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Rapid, Reproducible HPLC Analysis for Flavonoids in Cocoa

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


By Julie Kowalski, Restek Innovations Chemist, and Brian Shofran, LECO Corporation
  • 15-minute screening for flavonoids.
  • Excellent selectivity, using an Ultra Aqueous C18 column.
  • Reliable identifications and reproducible results for complex samples.

Flavonoids are complex polyphenolic compounds, with diverse aromatic substitutions, that contribute to color, flavor, fragrance—and toxicity—of many foods. Interest in flavonoids has exploded because of links to antioxidant activity and, possibly, to control and prevention of disease. [1,2]. 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 the flavan-3-ol flavonoids, including catechin, epicatechin, and procyanidin (Figure 1), and these are screened for as marker compounds. In finished chocolate and cocoa products, amounts of flavonoids depend primarily on the amounts of nonfat cocoa solids, on bean type, and on processing. Flavonoids can be destroyed by heat or other processing, like dutching, which is common in the production of cocoa and chocolate products.


Figure 1  Flavan-3-ol flavonoids are screened for as marker compounds.


We developed a rapid screening method for catechin, epicatechin, and procyanidin content, and screened commercial cocoa products for flavan-3-ol content. We prepared samples by mixing the cocoa products with liquid nitrogen, powdering the frozen mixes, and extracting samples with deionized water:methanol (1:4). Extracts were centrifuged, concentrated, and filtered [3]. For a detailed description of sample preparation, refer to the LECO website www.leco.com.

An Ultra Aqueous C18 column is an excellent choice for this analysis, because it is designed to perform reversed phase separations well and reproducibly when the mobile phase has a high aqueous content. Using a 100mm x 2.1mm Ultra Aqueous C18 column and the automated peak find LECO ChromaTOF software in the Unique LC-TOFMS system, we separated and identified 26 flavonoid compounds in a cacao sample (Figure 2 and Table 1).*


Figure 2  Extracted ion chromatogram of a cacao sample.


Sample:

 

Inj.:

5µL

Conc.:

500 mg sample extract

Sample diluent:

70% water/methanol

Autosampler temp:

10°C

Column:

Ultra Aqueous C18

Cat.# :

9178312

Dimensions:

100 x 2.1 mm

Particle size:

3µm

Pore size:

100Å

Conditions:

 

Mobile phase:

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

 

Time (min.)

%B

0

10

10

60

15

60

Flow:

400µL/min.

Temp.:

30°C

Det.:

UV @ 210nm

Mass Spectrometry

Instrument:

Leco Unique LC-TOFMS High Flow ESI Source

ESI voltage:

(-) 3500 V

Desolv. temp.:

300°C

Nebulizer pres.:

375kPa

Desolv. gas:

nitrogen, 7L/min.

Interface temp.:

100°C

Nozzle:

(-) 160V

Data acq. rate:

4 spectra/sec.

Numbered peaks are listed in Table 1


Table 1  Components in the cacao sample.
Peak
RT (min:sec)
Unique Mass
Area
Area %
1. unknown
00:35.5
273.0613
16924 
3.9
2. unknown
01:11.0
383.3092
1821
0.4
3. unknown
03:31.2
294.181
5757
1.3
4. catechin (monomer)
03:50.4
289.1818
28618  
6.7
5. unknown
04:07.7
369.1762
4530
1.1
6. unknown
04:14.4
305.1884
85897  
20.0  
7. procyanidin B2
04:24.0
577.3722
34559  
8.0
8. unknown
04:25.9
278.1767
4378
1.0
9. epicatechin
04:53.8
289.1841
93682 
21.8  
10. procyanidin C1
05:06.2
865.5671
10221 
2.4
11. procyanidin (tetramer)
05:17.8
1153.8179  
1585
0.4
12. clovamide
05:29.3
358.2409
3528
0.8
13. unknown
05:33.1
275.2085
6160
1.4
14. unknown
05:36.0
353.177
1586
0.4
15. procyanidin II-g
06:21.1
737.4785
5246
1.2
16. procyanidin B5
06:31.7
577.3745
10339  
2.4
17. procyanidin II-a
06:32.6
707.4643
4043
0.9
18. unknown
06:48.0
393.3242
2778
0.6
19. dideoxyclovamide
07:08.2
326.2384
4839
1.1
20. quercetin-galactoside
07:16.8
463.279
9471
2.2
21. quercetin-arabinoside
07:44.6
433.2524
9797
2.3
22. unknown
08:13.4
497.536
17417  
4.1
23. unknown
09:02.4
201.191
3097
0.7
24. quercetin
09:30.2
301.1595
2179
0.5
25. unknown
09:43.7
723.8071
52646  
12.3  
26. unknown
10:10.6
391.2756
8550
2.0

Next, using the automated peak find software in ChromaTOF, we identified flavonoids in cocoa powder (Figure 3 and Table 2). Processing of cacao reduces the amount of catechins and procyanidins in cocoa components. If an alkalizing step is present in the process, this also leads to a remarkable decrease in the content of catechins and procyanidins. 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 analysis was completed and conditions returned to the initial mobile phase composition in 15 minutes.


Figure 3  The flavonoid composition of cocoa powder is readily distinguished from that of cacao, using our column and detection system.


Sample:

 

Inj.:

5µL

Conc.:

500 mg sample extract

Sample diluent:

70% water/methanol

Autosampler temp:

10°C

Column:

Ultra Aqueous C18

Cat.# :

9178312

Dimensions:

100 x 2.1 mm

Particle size:

3µm

Pore size:

100Å

Conditions:

 

Mobile phase:

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

 

Time (min.)

%B

0

10

10

60

15

60

Flow:

400µL/min.

Temp.:

30°C

Det.:

UV @ 210nm

Mass Spectrometry

Instrument:

Leco Unique LC-TOFMS High Flow ESI Source

ESI voltage:

(-) 3500 V

Desolv. temp.:

300°C

Nebulizer pres.:

375kPa

Desolv. gas:

nitrogen, 7L/min.

Interface temp.:

100°C

Nozzle:

(-) 160V

Data acq. rate:

4 spectra/sec.

Numbered peaks are listed in Table 2

Table 2  Flavonoid components in cocoa powder exhibit virtually the same retention times as in cacao.
Peak
RT (min:sec)
Unique Mass
Area
Area %
1. Unknown
00:36.5
273.0620
16827 
4.2
2. Unknown
01:00.5
405.1844
3064
0.8
3. Unknown
01:20.6
283.1889
2835
0.7
4. Unknown
01:55.2
299.1908
3542
0.9
5. catechin
03:50.4
289.1806
35151 
8.7
6. Unknown
04:01.0
431.3734
4779
1.2
7. Unknown
04:12.5
305.1866
156954   
38.9  
8. Unknown
04:23.0
381.3214
6868
1.7
9. procyanidin B2
04:25.0
577.3661
3928
1.0
10. Unknown
04:35.5
381.3273
6601
1.6
11. epicatechin
04:52.8
289.1802
28030 
6.9
12. Unknown
05:19.7
333.1894
9199
2.3
13. clovamide
05:28.3
358.2432
3287
0.8
14. Unknown
05:32.2
275.2074
12865 
3.2
15. Unknown
06:08.6
333.1899
5070
1.3
16. Unknown
06:32.6
393.3275
9841
2.4
17. dideoxyclovamide
07:08.2
326.2279
7088
1.8
18. quercetin-galactoside
07:16.8
463.2485
6002
1.5
19. Unknown
07:37.9
516.4572
8285
2.1
20. quercetin-arabinoside
07:43.7
433.2532
6047
1.5
21. Unknown
08:13.4
497.5329
15347  
3.8
22. Unknown
09:43.7
723.8036
52001 
12.9  

Subsequently, we analyzed three samples from Venezuela, containing differing amounts of cacao. Quantitative results were determined through ChromaTOF. Analytical results for these samples are shown in Figure 4. As expected, based on data in Table 1, epicatechin was substantially higher than catechin in each sample. Also as expected, catechin, epicatechin, and procyanidin B2 content increased with increasing amounts of cacao.

A LECO Unique LC-TOFMS system and an Ultra Aqueous C18 column assure rapid, excellent resolution, reliable identification and quantification, and highly reproducible retention times for flavonoid compounds — even in very complex mixtures.


Figure 4  Concentrations of flavonoids in Venezuelan cacao, determined using an Ultra Aqueous C18 column and a LECO Unique LC-TOFMS system.

References

[1] Prior, R.L., et al., Procyanidin and catechin content and antioxidant capacity of cocoa and chocolate products, J. Agric. Food Chem. 54: 4057-4061 (2006).
[2] Hurst, W.J., et al., Antioxidant activity and polyphenols and procyanidin contents of selected commercially available cocoa-containing and chocolate products in the United States, J. Agric. Food Chem. 54: 4062-4068 (2006).
[3] Andreas-Lacueva, et al., An LC method for the analysis of cocoa phenolics, LC*GC Eur. 902-905 (2000).

Footnotes

  • *Cacao is the sum of the products derived from the cacao bean — chocolate liquor, cocoa, and cocoa butter(2).
  • LECO Corporation, 14950 Technology Court, Fort Myers, FL, 33912 54: 4062-4068 (2006).

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