Abstract
The analysis of phenolic compounds in wines is of considerable commercial importance, since they are known to play the major role in defying the sensorial characteristics of wines, such as astringency, flavor, and color, as well as in the browning process, causing product deterioration. In this paper, an automated reversed phase high performance liquid chromatographic method, using a multistep binary gradient elution, is developed for the determination of five phenolic acids: caffeic, ferulic, vanillic, salicylic, and p-hydroxy-benzoic acid. The separation method was based on mobile-phase optimization and off-line solid-phase extraction (SPE) from wines and wine vinegar samples, using novel sorbent materials.
The analytical column, an Inertsil C8, 250 × 4 mm, 5 μm, used, was operating at ambient temperature. The elution solvents were classified as A: 5–95 (v/v) CH3OH-H2O and B: 90−10 (v/v) CH3OH - H2O, both A and B acidified with glacial CH3COOH at pH 3–4. The samples were eluted with gradient starting with 10% in B and ending to 100% in B. Nicotinic acid was used as internal standard at a concentration of 10 ng/μL The flow rate of mobile phase was 0.8 mL/min and observed inlet pressure ranged from 270 to 345 kg/cm2. A diode-array detector monitored the effluent and chromatograms were recorded at 290 and 256 nm. Comparing their retention time values and UV spectra in the 190–300 nm range with authentic standards stored in a data bank, we made identification of phenolic compounds.
The statistical evaluation of the method was examined performing intra-day (n=8) repeatability and inter-day (n=8) precision assays at three concentration levels and was found to be satisfactory, with high accuracy and precision results. High percentage recoveries from wines (98.7 ± 5.9, with RSD 6.0) and wine vinegar (100.8 ± 6.4, with RSD 6.4) samples were achieved using Nexus SPE cartridges with hydrophilic and lipophilic properties and solvent B as eluent.