Coupling Hydrophilic Interaction Chromatography and Reverse-Phase Chromatography for Improved Direct Analysis of Grape Seed Proanthocyanidins

Abstract

Acid-catalyzed depolymerization is recognized as the most practical method for analyzing subunit composition and the polymerization degree of proanthocyanidins, involving purification by removing free flavan-3-ols, as well as acid-catalyzed cleavage and the identification of cleavage products. However, after the removal of proanthocyanidins with low molecular weights during purification, the formation of anthocyanidins from the extension subunits accompanying acid-catalyzed cleavage occurred. Thus, grape seed extract other than purified proanthocyanidins was applied to acid-catalyzed depolymerization. Hydrophilic interaction chromatography was developed to quantify free flavan-3-ols in grape seed extract to distinguish them from flavan-3-ols from terminal subunits of proanthocyanidins. Reverse-phase chromatography was used to analyze anthocyanidins and cleavage products at 550 and 280 nm, respectively. It is found that the defects of the recognized method did not influence the results of the subunit composition, but both altered the mean degree of polymerization. The established method was able to directly analyze proanthocyanidins in grape seed extract for higher accuracy and speed than the recognized method.

Keywords: anthocyanidin; direct analysis; hydrophilic interaction chromatography; mean degree of polymerization; proanthocyanidins.

Figure 1

Scheme of the recognized method reported previously and the direct method established in the present study for analyzing the structure of proanthocyanidins in grape seed extract.

Figure 2

Acid-catalyzed transformation of proanthocyanidins purified from grape seed extract into cyanidin. (A) Optical absorption spectra of cyanidin, proanthocyanidins in pure methanol, and proanthocyanidins after acid catalysis in the presence of excess phloroglucinol or not (insets are visual images of the methanolic solutions of proanthocyanidins). (B) HPLC chromatograms of cyanidin standard and proanthocyanidins after acid catalysis in the presence of excess phloroglucinol at 550 nm (inset is a mass spectrum of the peak detected at 48.29 min after acid catalysis).

Figure 3

Molecular weight distributions of starting proanthocyanidins, proanthocyanidins after acid-catalyzed cleavage in the absence of phloroglucinol, cyanidin, and catechin.

Figure 4

HPLC chromatograms of grape seed extract, removed polyphenols after purification of proanthocyanidins, and procyanidin B1, which were analyzed with HILIC based on their molecular weight.

Figure 5

HPLC chromatograms of grape seed extract, free flavan-3-ols, and procyanidin B1, which were analyzed with HILIC based on their molecular weight.

Figure 6

HPLC chromatograms of cleavage products from purified proanthocyanidins and grape seed extract following acid-catalysis in the presence of excess phloroglucinol. (1) catechin–phloroglucinol; (2) epicatechin–phloroglucinol; (3) catechin; (4) epicatechin gallate–phloroglucinol; (5) epicatechin; and (6) epicatechin gallate.

Figure 7

Comparison of the mDP analyzed with the recognized method (RM) and the direct method (DM), considering cyanidin formation or not. * p < 0.05; ** p < 0.01; *** p < 0.001.