Quantification of Podophyllotoxin From Podophyllum hexandrum Using HPLC-UV-DAD

Gradient RP-HPLC method for the quantification of podophyllotoxin from Podophyllum hexandrum has been developed. In our study, qualification of podophyllotoxin was performed by Thin Layer Chromatography (TLC) with R values of 0.85 (leaf) and 0.94 (root) f when compared with the standard. UV-VIS spectrophotometeric studies showed electronic absorption at γ_max value 284 nm. Gradient max chromatographic separation of Podophyllotoxin was performed on a 50 mm ×2.1 mm (i.d.) Macherey-Nagel NUCLEODUR C18 Gravity HPLC column, packed with 1.5 μm particles equipped with a 0.5m pre-filter (Upchurch Scientific, Oak Harbor, WA, USA), using mobile phase methanol/water (1/1, v/v) and 100% methanol, both containing 0.1% ammonium hydroxide (25%) and 10 mmol/L ammonium acetate (pH 9). Van Deemter Equation and Fundamental Resolution Equation were validated to measure optimum velocity of the mobile phase and to check resolution of the peaks. DAD detector was used to profile the exact compositions and to quantify podophyllotoxin herein. Chromatographic purity of podophyllotoxin was found to be 2.001% in leaf and 2.08% in root sample of Podophyllum hexandrum. Further, the information generated is useful for various stake holders like pharmaceutical industries, research institutes and others.