SPE-HPLC Determination of Chlorogenic and Phenolic Acids in Coffee

A new solid phase extraction-high-performance liquid chromatography method with ultraviolet detection was developed and validated for the determination of three chlorogenic acids (5-O-caffeoylquinic, 3-O-caffeoylquinic and 4-O-caffeoylquinic acids) and six phenolic acids (caffeic, ferulic, sinapic, protocatechuic, p-hydroxybenzoic acid (PHBA) and vanillic acid (VA)) in coffee bean samples. Extraction was performed using hydrophilic-lipophilic balance cartridges. Separations were accomplished using a C-18 guard column (10 x 2.1 mm, 3 mu m) and a C18 analytical column (50 x 2.1 mm, 3 mu m). o-Phosphoric acid solution (0.08%) and methanol/water/acetonitrile (85: 10: 5) solution were used as mobile phase with a gradient system. A UV detector was used at 325 nm for 5-O-caffeoylquinic, caffeic, 3-O-caffeoylquinic, 4-O-caffeoylquinic, ferulic, sinapic acids, and 215 nm for protocatechuic, PHBA and VA. Calibration equations and coefficients of determination were determined by least-squares method with weighting factor. Limits of detection and quantification were in the range of 0.15-0.69 and 0.46-2.09 mg/L, respectively. Precision and accuracy of the proposed method were investigated with coffee sample spiked at low, medium and high concentrations. The developed and validated method was applied for the determination of nine phenolic compounds in seven coffee bean samples from different origins with high accuracy and repeatability.