Development of a Simultaneous HPLC Determination Method of Cacao Polyphenols, Caffeine and Theobromine in High Cacao Chocolate by Core-Shell Type Reversed-Phase Columns

An isocratic and simple reversed-phase HPLC determination method of cacao polyphenols (CPPs) and methylxanthines in high cacao chocolate was developed with a core-shell (CS) type column and an extraction solvent of a mixture of methanol (MeOH) and acidic water. Simultaneous separation of four major CPPs, namely (-)-epicatechin (EC), (+)-catechin (C), procyanidin B2 and procyanidin C1, and two methylxanthines, caffeine (CAF) and theobromine (TBM) contained in chocolate was successfully performed within ca. 18 min. Among six CS type columns employed, a biphenyl type and a cholesteryl type column gave successful separation. A biphenyl type column, Velox Biphenyl (2.7 mu m, 4.6 mm x 100 mm) and the mobile phase of a mixture of acetonitrile and 0.1% (v/v) formic acid (10:90, v/v) were used for the assay of chocolate. A mixture of MeOH and 0.1% (v/v) formic acid (70:30, v/v) was effective for the extraction of six components from chocolate matrix, which contains various nutritional components such as fat, protein, sugar etc. More than 90% recoveries for CAF and TMB, and almost 100% recoveries for CPPs were obtained in validation studies. In the developed assay method, EC and CAF were used as the standard substance for six components considering cost limits and simplicity as a quality control method. Other than six components, one unknown peak eluted after CAF in the biphenyl column was identified by LC-MS/MS analysis. As a result, this was found as a fragrance ingredient, vanillin. Assay results by the developed HPLC method gave reasonable content values previously reported. Further determination of vanillin was possible together with assay of these six major components of chocolate.