HPTLC-Densitometry Method for Simultaneous Determination of Major Lignans and Flavonoids in Podophyllum hexandrum

A new quantitative method using thin-layer chromatography silica gel 60F(254) plates as the stationary phase and the mobile phase consisting of toluene-ethyl acetate-acetic acid (15.0:7.5:0.5, v/v) was employed for simultaneous determination of lignans and flavonoid in Podophyllum hexandrum. Densitometric determination of the constituents was performed at 254 nm in reflectance/absorbance mode. The method was validated for precision, accuracy, specificity, robustness, and recovery. The linear regression analysis data for the calibration plots showed a good linear relationship (r(2) = 0.9903-0.9976) in the calibration range of 1-8 mu g per band for 4'-O-demethylpodophyllotoxin (1), podophyllotoxin (2), and podophyllotoxone (4), and 2-10 mu g per band for kaempferol (3) and deoxypodophyllotoxin (5) with respect to peak area. Limits of detection and quantitation were in the range of 250-617 ng per band and 856-1974 ng per band. The average recovery for 4'-O-demethylpodophyllotoxin, podophyllotoxin, kaempferol, podophyllotoxone, and deoxypodophyllotoxin was 96.38 +/- 1.92 to 101.84 (+/-) 1.05%, indicating the excellent reproducibility. Podophyllotoxin was found in highest content (9.92 mu g mg(-1)) and podophyllotoxone in the lowest content (0.94 mu g mg(-1)). The proposed method is rapid, simple, precise, specific, sensitive, accurate, and robust.