A novel supercritical fluid chromatography - evaporative light scattering detection method for the quantification of paraconic acids in European Cetraria islandica (Iceland moss) samples.

Paraconic acids are bioactive constituents of Iceland moss, a medicinal drug widely used to relieve coughs. They are characterized by high lipophilicity and a γ-butyrolactone ring system but lack strong UV-vis chromophores. Consequently, LC-DAD, despite remaining one of the most widely used analytical techniques in natural products research, is not suitable for their analysis. To overcome this limitation, we developed and validated a novel supercritical fluid chromatography (SFC) method for the rapid analysis of paraconic acids in Cetraria islandica. It successfully resolved four relevant derivatives in Iceland moss samples within a separation time of under 8 min. Optimal separation was achieved in gradient mode using a Viridis BEH-2EP column with supercritical carbon dioxide (CO2) as the primary mobile phase and a modifier consisting of 2.5% acetic acid in a methanol/acetonitrile mixture (6:4). Detection and quantification were achieved with an evaporative light-scattering detector (ELSD). All determined validation parameters were in accordance with International Council for Harmonization (ICH) guidelines, and the method's practical applicability was demonstrated by analyzing different C. islandica samples. Their paraconic acid profile varied both qualitatively and quantitatively, e.g., (±)-protolichesterinic acid ranged from 0.53 to 5.11 mg/g. The developed method offers a favorable combination of excellent analytical performance, sustainability, and practicality, as confirmed by a high whiteness parameter value of 73.3 (White Analytical Chemistry (WAC) index).