TLC and HPLC Screening of p-Coumaric Acid, frans-Resveratrol, and Pterostilbene in Bacterial Cultures, Food Supplements, and Wine

摘要

A thin-layer chromatographic (TLC) method for fast screening of Ira/ts-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 /rans-resvera-trol 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 fra/w-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 x 4.6 mm I.D., particle size: 5 u.m) 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 mL min"1.