RP-UHPLC and HPTLC Method Development and Validation for Analysis of Andrographolide from Herbal Hepatoprotective Formulation

Abstract

This work aimed to create a speedy, precise, and selective HPTLC and RP-UHPLC method for analysing andrographolide in fi nished products and raw materials. Asian medicines are using Andrographis paniculata since long. It’s used to treat skin eruptions, boils, scabies, and chronic, unexplained fevers, all of which are caused by blood “abnormalities.” Liquid chromatographic methods were developed to separate andrographolide and its herbal dosage form. HPTLC chromatography employed a 10 by 10 cm aluminum plate coated with 0.2 mm of silica gel 60 F254 (E. Merck, Germany). Camag Linomat 5 applicator with 100 μL syringe was used to apply samples in 6 mm bands (Hamilton, Switzerland). 14 mm separated the two bands, and 150 nL sec-1 was applied. Mobile phase was dichloromethane, toluene, ethyl acetate, and formic acid (6:4:1:0.5). This chromatogram runs 80 mm. Camag TLC scanner measured density at 254 nm. Mean RF=0.69. The linear calibration curve covers 500–3000 ng/spot and has a 0.996 correlation coeffi cient. Limit of Detection: 31.5 ng; Limit of Quantitation: 95.48 ng. A validated RP-UHPLC method for quantifying andrographolide in extract and formulation has been established. UHPLC analysis was performed in isocratic mode, at room temperature, using acetonitrile: Water (0.2% acetic acid) (85:15, v/v) as mobile phase on a 250 mm 4.6 mm i.d., 5 μm Cosmosil C18 column. Detection was at 230 nm. Andrographolide has a 4.1 minute half-life. Between 10 and 60 g/mL, andrographolide was linear. The approach met or exceeded ICH’s linearity, precision, accuracy, and robustness