Abstract
Homogeneous liquid⁻liquid extraction (HLLE) has attracted considerable interest in the sample preparation of multi-analyte analysis. In this study, HLLEs of multiple phenolic compounds in propolis, a polyphenol-enriched resinous substance collected by honeybees, were performed for improving the understanding of the differences in partition efficiencies in four acetonitrile⁻water-based HLLE methods, including salting-out assisted liquid⁻liquid extraction (SALLE), sugaring-out assisted liquid⁻liquid extraction (SULLE), hydrophobic-solvent assisted liquid⁻liquid extraction (HSLLE), and subzero-temperature assisted liquid⁻liquid extraction (STLLE). Phenolic compounds were separated in reversed-phase HPLC, and the partition efficiencies in different experimental conditions were evaluated. Results showed that less-polar phenolic compounds (kaempferol and caffeic acid phenethyl ester) were highly efficiently partitioned into the upper acetonitrile (ACN) phase in all four HLLE methods. For more-polar phenolic compounds (caffeic acid, p-coumaric acid, isoferulic acid, dimethoxycinnamic acid, and cinnamic acid), increasing the concentration of ACN in the ACN⁻H₂O mixture could dramatically improve the partition efficiency. Moreover, results indicated that NaCl-based SALLE, HSLLE, and STLLE with ACN concentrations of 50:50 (ACN:H₂O, v/v) could be used for the selective extraction of low-polarity phenolic compounds. MgSO₄-based SALLE in the 50:50 ACN⁻H₂O mixture (ACN:H₂O, v/v) and the NaCl-based SALLE, SULLE, and STLLE with ACN concentrations of 70:30 (ACN:H₂O, v/v) could be used as general extraction methods for multiple phenolic compounds.