Fast and improved separation of major coumarins in Ammi visnaga (L.) Lam. by supercritical fluid chromatography

The first supercritical fluid chromatography method for the determination of five major coumarins (dihydrosamidin, visnadin, samidin, khellin, and visnagin) in Ammi visnaga fruits is described. Their baseline separation was possible in less than 5 min by using a UPC² HSS C₁₈ SB column with 1.8 μm particle size and a mobile phase comprising CO₂ , methanol, acetonitrile, and diethylamine. The type of stationary phase used was of particular relevance because, except for the selected one, the others did not resolve the two structural isomers dihydrosamidin and visnadin. Method validation confirmed that the procedure is linear (R² ≥ 0.9996) in a concentration range from 6 to 480 μg/mL, it is accurate (recovery rates: 97.2-103.6%) and precise (intraday deviation ≤ 6.6%, intraday deviation ≤ 1.7%); injecting 1 μL of standard solution, the determined limit of detection was below 1.9 μg/mL for all compounds. The analysis of different A. visnaga samples revealed their similar compositions, and khellin (0.75-1.01%) and visnagin (0.18-0.46%) were the dominant coumarins. Visnadin and dihydrosamidin, the individual quantification of which is described for the first time, were present at concentrations below 0.14%.