A thin-layer chromatographic (TLC) method for fast screening of trans-resveratrol, pterostilbene, and p-coumaric acid in samples of recombinant bacterial cultures, food supplements, and wine was developed. The separation was performed on high-performance thin-layer chromatography (HPTLC) silica gel 60 plates using n-hexane-ethyl acetate-formic acid (20:19:1, v/v) as developing solvent in tank configuration of horizontal developing chamber, in which better resolution between trans-resveratrol and p-coumaric acid than in sandwich configuration of the same chamber or in automatic developing chamber (ADC) was obtained. Compounds were detected before and after post-chromatographic derivatization (three detection reagents) by image analyzing system (at 366 nm or white light) and by densitometer (absorption-reflectance and fluorescence mode). The lowest densitometric limits of detection (LOD) 2 ng for trans-resveratrol (303 nm), 5 ng for pterostilbene (303 nm), and 15 ng for p-coumaric acid (286 nm) were found before derivatization in absorption-reflectance mode. Post-chromatographic derivatization with anisaldehyde-sulfuric acid detection reagent resulted in higher LOD in the same mode: 13 ng for trans-resveratrol and pterostilbene at 500 nm and 30 ng for p-coumaric acid at 566 nm. Natural fluorescence of both stilbenes was less sensitive than UV absorption and less selective than post-chromatographic derivatization with anisaldehyde reagent at densitometric screening of trans-resveratrol in the samples. A complementary high-performance liquid chromatography (HPLC) method was developed for screening and quantification of the three compounds in recombinant bacterial cultures. Adequate separation of the analytes was performed in 35 min by a gradient elution from a stainless-steel column Hypersil ODS (150 × 4.6 mm I.D., particle size: 5 μm) with the mobile phase consisting of 50 mM sodium acetate buffer pH 5.6 (solvent A) and acetonitrile (solvent B) at the flow rate of 1.5 mLmin−1.
L. Schmidlin, A. Poutaraud, P. Claudel, P. Mestre, E. Prado, M. Santos-Rosa, S. Wiedemann-Merdinoglu, F. Karst, D. Merdinoglu, P. Hugueney, Plant Physiol. 148 (2008) 1630–1639.
R.J. Conrado, G.C. Wu, J.T. Boock, H. Xu, S.Y. Chen, T. Lebar, J. Turnšek, N. Tomši M. Avbelj, R. Gaber, T. Koprivnjak, J. Mori, V. Glavnik, I. Vovk, M. Benèina, V. Hodnik, G. Anderluh, J.E. Dueber, R. Jerala, M.P. DeLisa, Nucleic Acids Res. (2011) 1–11, doi:10.1093/nar/gkr888.
S. Malovaná, F.J.G. Montelongo, J.P. Pérez, M.A. Rodríguez-Delgado, Anal. Chim. Acta 428 (2001) 245–253.
H. Jork, W. Funk, W. Fischer, H. Wimmer, Dünnschicht-Chromatographie: Reagenzien und Nachweismethoden, Bd. 1a, Physikalishe und chemische Nachweismethoden: Grundlagen, reagenzien I. VCH Verlagsgesellschaft, Weinheim, 1989.
Wimmer H., '', in Dünnschicht-Chromatographie: Reagenzien und Nachweismethoden, Bd. 1a, Physikalishe und chemische Nachweismethoden: Grundlagen, reagenzien I, (1989) -.
Wimmer H.Dünnschicht-Chromatographie: Reagenzien und Nachweismethoden, Bd. 1a, Physikalishe und chemische Nachweismethoden: Grundlagen, reagenzien I1989)| false