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

J Chromatogr A. 2010 Jan 29;1217(5):642-7. doi: 10.1016/j.chroma.2009.11.097. Epub 2009 Dec 4.


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 1.8ng/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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Beverages / analysis
  • Chromatography, High Pressure Liquid / methods*
  • Citrus / chemistry
  • Electrochemical Techniques / methods*
  • Flavones / analysis*
  • Flavonoids / analysis
  • Hydrogen-Ion Concentration
  • Linear Models
  • Reproducibility of Results
  • Sensitivity and Specificity


  • Flavones
  • Flavonoids
  • 5-hydroxy-3,6,7,8,3',4'-hexamethoxyflavone
  • salvigenin
  • umuhengerin