Simultaneous determination of four 5-hydroxy polymethoxyflavones by reversed-phase high performance liquid chromatography with electrochemical detection

Accumulating evidence has suggested the potential health-promoting effects of 5-hydroxy polymethoxyflavones (5-OH-PMFs) naturally existing in citrus genus. However, research efforts are hampered by the lack of reliable and sensitive methods for their determination in plant materials and biological samples. Using reversed-phase high performance liquid chromatography (HPLC) equipped with electrochemical (EC) detection, we have developed a fast and highly sensitive method for quantification of four 5-OH-PMFs, namely 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone, 5-hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone, 5-hydroxy-6,7,4′-trimethoxyflavone, and 5-hydroxy-6,7,8,4′-tetramethoxyflavone. The method was fully validated in terms of linearity, accuracy and precision. The limit of detection (LOD) was determined as being between 0.65 and 1.8 ng/mL (ppb), demonstrating an over 160 times higher sensitivity in comparison with the previously reported method using UV detection. The recovery rate of the method was between 96.17% and 110.82%, and the precision for the retention times and peak areas was all below 13%. The method was successfully used to quantify 5-OH-PMFs with a wide range of abundance in the citrus products and preparations, such as orange juice, citrus peel, and dried tangerine peel. The quantification method for 5-OH-PMFs developed herein could be useful for the nutritional and pharmacological studies of these compounds in future.