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 1Flavan-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 2Extracted 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.# : RE9178312 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

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 3The 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.# : RE9178312 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

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 4Concentrations 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).