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Validation of the TLC Method for Quantification of (-)-Epicatechin and Procyanidin B2 in Chocolate

Monomeric flavan-3-ols such as catechin, epicatechin (EC), gallocatechin and epigallocatechin and oligomeric compounds derived from them, named proanthocyanidins, are beneficial for human health [1]. They occur in various types of fruits, nuts, spices and also in beans of cocoa (Theobroma cacao L.) consequently also in chocolate. Chocolate contains EC as the main flavan-3-ol and different procyanidins [2]. The goal of the present study was validation of our miniaturized TLC method for densitometric quantification of the two major phenolic compounds, procyanidin B2 (B2) and its monomeric unit EC, in chocolate. Test solutions of chocolate were obtained in 80% aqueous acetone. The validation procedure anticipated HPTLC cellulose plate and 1-propanol-water-acetic acid (20:80:1, v/v/v) [3] as developing solvent. For detection, our newly developed derivatisation reagent [4] with 4-dimethylaminocinnamaldehyde was used. Recovery (90 – 100%) and repeatability (RSD<10%) of the method satisfied the prescribed criteria for both compounds. Detection of both compounds with the proposed reagent was specific and sensitive with the limit of detection 0.2 ng for EC and 2 ng for B2. The light blue colouring of the lane, because of the presence of polymeric procyanidins in chocolate, could be subtracted at densitometric evaluation without difficulty. Polynomial curves (2-30 ng for EC and 4-60 ng for B2) were obtained for quantification of both analysed compounds. Plates with the applications of EC and B2 standards should be developed immediately after application. After derivatisation, densitometric evaluation should be performed within 90 min. Standards of EC and B2 were stable at least 7 days stored at + 4 °C, -20 °C or -80 °C in stock (500 μ g EC/ml and 100 μ g B2/ml) as well as in diluted solutions in methanol (1 μ g/ml to 10 μ g/ml of EC and B2). The method can be applied for the routine quality control of the two major phenolic compounds present in different chocolate samples. References: [1] T. Kurosawa, F. Itoh, A. Nozaki, Y. Nakano, S.-I. Katsuda, N. Osakabe, H. Tsubone, K. Kondo, H. Itakura, Atherosclerosis 179 (2005) 237-246. [2] L.W. Gu, M.A. Kelm, J.F. Hammerstone, G. Beecher, J. Holden, D. Haytowitz, S. Gebhardt, R.L. Prior, J. Nutr. 134 (2004) 613-617. [3] I. Vovk, B. Simonovska, H. Vuorela, J. Chromatogr. A 1077 (2005) 188-194. [4] V. Glavnik, B. Simonovska, I. Vovk. J. Chromatogr. A (2008) submitted.

validation; chocolate; (-)-epicatechin; procyanidin B2; thin-layer chromatography

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