Simultaneous Detection of Protocatechuic Acid, Chlorogenic Acid and Caffeic Acid in Honey by HPLC with Ultraviolet and Electrochemical Detectors

A rapid and sensitive method for the detection of protocatechuic acid (PA), chlorogenic acid (CLA) and caffeic acid (CAA) has been developed by high-performance liquid chromatography (HPLC) with an ultraviolet detector (UV) and an electrochemical detector (ECD). HPLC with UV detection was performed on a C18 column (250×4.6 mm, 5 μm) with methanol (pH 4.0) - acetic acid (v/v, 85/15) as the mobile phase at a flow rate of 1.0 mL/min. ECD detection was performed on a three-electrode system, with a glassy carbon electrode, a platinum foil and a saturated calomel electrode (SCE) as the working electrode, the auxiliary electrode and the reference electrode, respectively. Compared with individual HPLC-UV and ECD methods, the presented HPLC (UV-ECD) technique had the advantage of enhanced capture of electroactive PA, CLA and CAA and achieved obvious detection sensitivity. Under the optimized conditions, the linear concentration ranges were from 0.0005 to 6 mg L-1 for PA and CAA, and from 0.0015 to 18 mg L-1 for CLA, with limits of detection of 10 ng L-1 for PA, 30 ng L-1 for CLA and 13 ng L-1 for CAA (S/N = 3), which were clearly lower than those obtained with the HPLC-UV method. The approach was also successfully applied to detection in honey samples with satisfactory recoveries.