(-)-Catechin in cocoa and chocolate: occurrence and analysis of an atypical flavan-3-ol enantiomer

Abstract

Cocoa contains high levels of different flavonoids. In the present study, the enantioseparation of catechin and epicatechin in cocoa and cocoa products by chiral capillary electrophoresis (CCE) was performed. A baseline separation of the catechin and epicatechin enantiomers was achieved by using 0.1 mol x L(-1) borate buffer (pH 8.5) with 12 mmol x L(-1) (2-hydroxypropyl)-gamma-cyclodextrin as chiral selector, a fused-silica capillary with 50 cm effective length (75 microm I.D.), +18 kV applied voltage, a temperature of 20 degrees C and direct UV detection at 280 nm. To avoid comigration or coelution of other similar substances, the flavan-3-ols were isolated and purified using polyamide-solid-phase-extraction and LC-MS analysis. As expected, we found (-)-epicatechin and (+)-catechin in unfermented, dried, unroasted cocoa beans. In contrast, roasted cocoa beans and cocoa products additionally contained the atypical flavan-3-ol (-)-catechin. This is generally formed during the manufacturing process by an epimerization which converts (-)-epicatechin to its epimer (-)-catechin. High temperatures during the cocoa bean roasting process and particularly the alkalization of the cocoa powder are the main factors inducing the epimerization reaction. In addition to the analysis of cocoa and cocoa products, peak ratios were calculated for a better differentiation of the cocoa products.

Figure 1

Structures of the catechin /epicatechin enantiomers and procyanidin B2.

Figure 2

Electropherogram showing the enantioselective separation of catechin and epicatechin in a) an extract of unfermented, dried, unroasted cocoa beans and b) isolated from this extract. 1: (-)-Epicatechin, 2: (+)-Catechin, P: Procyanidin B2. Experimental conditions: uncoated fused-silica capillary [60 cm (effective length 50 cm) × 75 µm I.D.]; electrolyte: 0.1 mol·L^-1 borate buffer, pH 8.5; chiral selector: 12.0 mmol·L^-1 HP-γ-CD; voltage: 18 kV; UV-Detection: 280 nm; temperature: 20 °C; injection: pressure, 3 s, 0.3 p.s.i..

Figure 3

Electropherogram showing the enantioselective separation of catechin and epicatechin in a) an extract of roasted cocoa beans and b) isolated from this extract. 1: (-)-Epicatechin, 2: (+)-Catechin, 3: (-)-Catechin, P: Procyanidin B2. Experimental conditions as in Figure 2.

Figure 4

Electropherogram showing the enantioselective separation of catechin and epicatechin in a) an extract of dark chocolate and b) isolated from this extract. 1: (-)-Epicatechin, 2: (+)-Catechin, 3: (-)-Catechin, P: Procyanidin B2. Experimental conditions as in Figure 2.

Figure 5

Electropherogram showing the enantioselective separation of catechin and epicatechin in a) an extract of cocoa powder and b) isolated from this extract. 1: (-)-Epicatechin, 2: (+)-Catechin, 3: (-)-Catechin, P: Procyanidin B2. Experimental conditions as in Figure 2.

Figure 6

MS spectra (averaged over the retention time frame of the flavan-3-ols, 20-35 min) of a) the raw cocoa bean extract and b) the isolated and purified flavan-3-ols from this extract. The signals in the extract spectrum correspond to procyanidins. The small signals in the isolated flavanols correspond to ionization-induced adducts and fragments and background noise.