Simultaneous determination of four andrographolides in Andrographis paniculata Nees by silver ion reversed-phase high-performance liquid chromatography.

A rapid and sensitive silver ion high-performance liquid chromatographic (Ag[I]-HPLC) method is developed for the simultaneous determination of the biologically active diterpenoids andrographolide, 14-deoxy-11,12-didehydroandrographolide, 14-deoxyandrographolide, and neoandrographolide in Andrographis paniculata Nees. HPLC is carried out for determining andrographolide and its derivatives with methanol-water (55:45, v/v) as the mobile phase on a C18 column (5 microm, 150 mm x 4.6 mm i.d.) with UV detection at 205 nm. Four andrographolides are baseline separated in a novel way: by adding silver ions (0.005 mol L(-1)) to the previously mentioned mobile phase. Validation of the method challenges specificity, linearity, limit of detection, limit of quantitation, accuracy, and repeatability, and the results met the acceptance criteria for all analytes. The molecular mechanism of retention is demonstrated by comparing partition coefficients (logP) of different andrographolides and andrographolide-Ag(I) complexes. Thus, the method is successfully applied to characterize and determine the four andrographolides in Andrographis paniculata Nees extract and its commercial product.