Separation of flavonoids by semi-micro high-performance liquid chromatography with electrochemical detection

A highly sensitive and selective method was developed for the determination of 15 flavonoids by high-performance liquid chromatography with electrochemical detection (HPLC-ECD) using a microbore column. The 15 flavonoids were divided into two groups according to their hydrophobicity and were resolved by two isocratic systems, namely, methanol-water (1:1 and 3:7, v/v) containing 0.5% phosphoric acid. The retention factor (k) of each flavonoid linearly correlated with the logarithmic partition of coefficients between 1-octanol-water (log P) values. The detection limits (S/N = 3) of the flavonoids tested were in the range of 2-25 fmol; that is 600 times more sensitive than conventional HPLC with UV detection. The relative standard deviations (RSD, n = 5) were less than 5% at 1 pmol for the flavonoids tested. By the present method, only 100 muL of bottled Japanese green tea were enough to determine the flavonoid contents.