Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05–5.0 μg mL−1. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2–6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL−1 for andrographolide and 0.022 μg mL−1 for dehydroandrographolide. This method was successfully applied to the plasma concentration–time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.