COLORIMETRIC METHOD FOR THE DETERMINATION OF HECOGENIN IN AGAVE AMERICANA L. RESIDUE

A method has been developed for the determination of hecogenin content in Agave americana L. residue after separation of fibers. It includes extraction, separation and determination of hecogenin, these operations are as follows:(1) Weigh accurately 0.2～0.3g of pulverived sample. Macerate it in a flask with 10 ml of 10% H₂SO₄ solution, heat for 30 minutes on an electric hot plate. After cooling, filter the residue with G₄ sintered glass funnel and wash the residue with water until the eluate is not acidic. Dry the residue in an electric oven for 2 hours, then wash it with CHCl₃—MeOH (1:1) into a 10 ml volumetric flask.(2) Take exactly 50 μl of the solution and chromatograph on a silica gel G plate with CH₂Cl₂—MeOH (95:5) as the developing solvent. After drying, the spots are detected by iodine vapor. Scrape the hecogenin spot from the plate into a small column, wash with CH₂Cl₂—MeOH (1:1), collect 1～2 ml of eluate and evaporate to dryness.(3) Treat the residue with 0.1 ml 5% vanillin reagent and 0.2 ml HClO₄ and keep at 70℃ for 30 min. Cool and dilute to 4.0 ml with glacial acetic acid, measure the absorbance at 540 nm against a reagent blank and calculate the amoun of hecogenin from a calibration curve.