Reversed-phase high-performance liquid chromatography determination of selected phenolic acids in propolis concentrates in terms of standardization for drug manufacturing purposes

• Published in: Journal of AOAC International Vol. 89; no. 2; pp. 352 - 364
• Main Authors: Krzek, J, Kaleta, J, Hubicka, U, Niedzwiedz, A
• Format: Journal Article
• Language: English
• Published: England 01.03.2006
• Subjects: active ingredients; Anti-Bacterial Agents; antibacterial properties; Bacteria - drug effects; caffeic acid; Chromatography, High Pressure Liquid; combs (social insects); concentrates; Drug Compounding; drug formulations; Drug Industry - standards; ferulic acid; Hydroxybenzoates - analysis; Indicators and Reagents; Microbial Sensitivity Tests; minimum inhibitory concentration; p-coumaric acid; propolis; Propolis - analysis; Propolis - pharmacology; Quality Control; Reference Standards; Reproducibility of Results; reversed-phase high performance liquid chromatography; solid phase extraction
• ISSN: 1060-3271; 1944-7922
• DOI: 10.1093/jaoac/89.2.352

A reversed-phase high-performance liquid chromatography method with gradient elution was developed for the determination of the caffeic, p-coumaric, and ferulic acids in propolis concentrates. Solid-phase extraction on an RP18 column was applied for preliminary purification, and chromatographic separation was performed on 100 RP18e Lichrospher column of particle size 5 micrometer. The mobile phase was obtained by mixing in appropriate ratios 0.03 mM NaH2PO4, acidified with H3PO4 up to pH = 3.0, with acetonitrile to obtain a gradient in the elution process. Spectrophotometric detection was conducted at 320 nm. Under the established conditions, the method featured high sensitivity, good precision, and comparability of results, as proven by method validation and statistical analysis of the obtained results. The limits of detection were 0.315, 0.325, and 0.695 microgram/mL for caffeic, p-coumaric, and ferulic acids, respectively. The corresponding recovery values were 98.14, 101.05, and 99.42% and the linearity ranges from 1.31 to 99.18 microgram/mL, 1.52 to 119.16 microgram/mL, and 2.42 to 184.14 microgram/mL. The precision of the method was expressed by relative standard deviation values that did not exceed 3%. It was also shown that the propolis concentrates under examination had similar antibacterial activity against Staphylococcus aureus ranging from 119.8 to 124.3 microgram/mL, contrary to model mixtures that showed no antibacterial activity.