Determination of Organic Acids in Propolis by HPLC Using Two Columns with an On-Line SPE System

Abstract

The HPLC method consisting of two columns and an on-line SPE system was developed for analysis of propolis extracts from Slovakia. The IEC column with spectrophotometric detection was tested for the separation of acids of the “shikimate pathway” and the C₁₈ column with on-line spectrophotometric (chlorogenic, rosmarinic, p-hydroxybenzoic acids) and fluorimetric (p-hydroxybenzoic acid) detection was tested for separation and determination acids in the water extract of propolis. For the preconcentration of compounds the on-line SPE on the C₁₈ preseparation guard column was used. The limits of determination were 0.2 µg · mL⁻¹ for shikimic acid, 20 µg · mL⁻¹ for quinic acid, 0.3 µg · mL⁻¹ for chlorogenic acid, 0.5 µg · mL⁻¹ for rosmarinic acid, 0.3 µg · mL⁻¹for p-hydroxybenzoic acid (UV), and 2 µg · mL⁻¹ for p-hydroxybenzoic acid (FL). On the basis of chromatographic characteristics and optical properties (UV spectra) chlorogenic acid, quinic acid, and shikimic acid were characterized in tested samples of propolis. The p-hydroxybenzoic acid could not be determined in the propolis extract because the interferences of unknown compounds with the same retention factor occur.

Keywords:

• Column liquid chromatography
• Propolis
• Organic acids

ACKNOWLEDGMENT

Work was supported by VEGA grants no. 1/0058/08 and no. 1/4291/07 and APVV project 20–035205.

Notes

^a Made in six replicates.

^b Made in three replicates.

^c Three samples injected three times each.

^d UV 330 nm.

^e UV 255 nm.

^f FL λ_Exc 265 nm λ_Em 350 nm.

^g UV 210 nm.