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

A new quantitative method using thin-layer chromatography silica gel 60F254 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 (r2 = 0.9903–0.9976) in the calibration range of 1–8 µg per band for 4′-O-demethylpodophyllotoxin (1), podophyllotoxin (2), and podophyllotoxone (4), and 2–10 µg 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 µg mg−1) and podophyllotoxone in the lowest content (0.94 µg mg−1). The proposed method is rapid, simple, precise, specific, sensitive, accurate, and robust.